Download Redox regulation of cysteine

Published December 4, 2014
Redox regulation of cysteine-dependent enzymes1
R. P. Guttmann2
Departments of Gerontology and Physiology, and the Sanders-Brown Center on Aging,
University of Kentucky, Lexington 40536
ABSTRACT: It is well-established that maintenance
of the intracellular redox (i.e., reduction-oxidation)
state is critical for cell survival and that prolonged or
abnormal perturbations toward oxidation result in cell
dysfunction. This is exemplified by the widespread observation of oxidative stress in many pathological conditions, as well as the positive effects of antioxidants in
treating certain conditions or extending the life span
itself. In addition to the effects of oxidation on the lipid
bilayer and modification of DNA in the nucleus, proteins are also modulated by the redox state. One of
the primary targets of oxidation within a protein is the
AA cysteine, whose thiol side chain is highly sensitive
to all types of oxidizing agents. Although this sensitivity is used to prevent oxidation within the cell by
potent defense mechanisms, such as glutathione, the
use of cysteine in the active site of enzymes leaves them
open to oxidant-mediated damage. Whether the damage is due to a pathological condition or to postmortem
mediated loss of redox homeostasis, cysteine-dependent
enzymes are targets of all forms of reactive oxygen,
nitrogen, and sulfur species. A greater understanding
of the redox-mediated control of cysteine-dependent
enzymes opens the door to the selective use of antioxidants to prevent or reverse the cellular damage their
inhibition causes.
Key words: aging, antioxidant, diet, methionine, oxidative stress, postmortem
©2010 American Society of Animal Science. All rights reserved.
INTRODUCTION
In addition to many well-known posttranslational
modifications, such as phosphorylation, that regulate
protein function, modifications mediated by reductionoxidation (redox) are also recognized as a significant
source of enzymatic control with general importance
in normal cell function (Janssen-Heininger et al., 2008;
Ottaviano et al., 2008; Trachootham et al., 2008; Bonham and Vacratsis, 2009; Figtree et al., 2009; Pervaiz
et al., 2009). Primarily, protein sensitivity to oxidation
is mediated by a reactive sulfur species within either
methionine or cysteine, although other side chains are
also susceptible. Of relevance here, enzymes that utilize
cysteine either as an important structural component
or within their active site are often discovered to be
regulated by the redox state of their environment (Bon-
1
Based on a presentation at the Cell Biology Symposium titled
“Redox regulation of cell function” at the Joint Annual Meeting,
July 12 to 16, 2009, Montreal, Canada. Manuscript publication
sponsored by the Journal of Animal Science and the American Society of Animal Science. Supported by National Institutes of Health
grants AG026521 and AG05144.
2
Corresponding author: [email protected]
Received August 7, 2009.
Accepted September 29, 2009.
J. Anim. Sci. 2010. 88:1297–1306
doi:10.2527/jas.2009-2381
ham and Vacratsis, 2009; Figtree et al., 2009; Leonard
et al., 2009; Nadeau et al., 2009). Changes in the redox
microenvironment occur because of various reactive
oxygen, nitrogen, and sulfur species as well as because
of modifications by cysteine-containing proteins or peptides. Although pathological states are easiest to understand with regard to redox imbalance, increased reactive species are generated under physiological states as
well.
Physiologically, thiol-reactive species are generated
through several mechanisms. One prominent means by
which reactive species are created is as a by-product of
energy production in mitochondria that produces the
oxidant superoxide. Oxidases, such as xanthine oxidase,
in addition to receptor-mediated peroxide production,
such as through the insulin receptor, are other examples
of physiological pathways that alter the redox balance.
Pathologically, increased oxidative stress has been associated with many conditions because loss of cellular
membrane integrity allows the highly oxidizing extracellular compartment to permeate. Although hotly debated, there remains good evidence supporting a positive role for prevention of oxidative stress (Parkes et al.,
1998; Ferreira and Reid, 2008; Jang et al., 2009; Perez
et al., 2009; Zhang et al., 2009), and maintenance of
cysteine homeostasis is likely to be vital in this regard.
For example, as discussed subsequently, many key en-
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zymes linked to transcription, energy production, apoptosis, and general cell signaling have critical cysteines
in their active sites, which, once oxidized, result in cellular dysfunction.
In addition to endogenous production of oxidants,
changes in redox are also produced by exogenous sources, such as by the use of various interventions used to
treat animals. Either by design or because of a side
effect, treatments to prevent or cure disease in animals
may have a significant impact on the balance of oxidants and antioxidants. Obviously, mitochondria are
one such drug target that can have a dramatic impact
on the production of oxidants because of the oxidizing
chemistry involved in energy production.
Finally, understanding the potential effects of thiol
oxidation is significant because of its importance in
the production and storage of quality food products.
It is well known that postmortem changes result in increased oxidation, leading to decreased initial quality
as well as spoilage of food (Koohmaraie, 1992; Boehm
et al., 1998; Chen et al., 2008). Because of the highly
sensitive nature of the thiol group within cysteine, an
understanding of the role of oxidation of key cysteinedependent enzymes will assist researchers in developing
improved techniques to combat the undesired outcomes
of such oxidation-mediated damage more effectively.
REDOX HOMEOSTASIS
Redox refers to the loss or gain of electrons, resulting
in a change in the oxidation number of an atom. This
review focuses on redox changes with respect to the AA
cysteine, because cysteine is one of the major sources
of redox balance in biological systems (Claiborne et al.,
2001; Barford, 2004; Jacob et al., 2006; Jones, 2008).
This includes a variety of cysteine-dependent enzymatic
and nonenzymatic constituents as parts of an intricate
system that works to maintain redox homeostasis.
Homeostasis is a critical factor in the long-term survival of a cell, and all cellular systems function with a
balance of constituents that are needed to work together. There are times, however, when that balance must
shift, such as when kinase activity in a cell is increased
to support specific functions. Because prolonged increases in kinase activity would likely lead to cellular
dysfunction, this increase cannot last and homeostasis
is eventually restored by phosphatase activity. Thus,
although homeostatic set points may change with time
or events, there is always a balance to be reached, although this may be arguable in some instances, such
as in the case of normal aging mechanisms, which may
purposefully move in one direction or another with time.
However, similar to the kinase-phosphatase system, redox regulation is maintained by an array of oxidant and
antioxidant systems working together to achieve proper
balance of needed oxidation for respiration, signaling,
and reducing potential to prevent undesirable oxidation
of cysteine-dependent enzymes or other redox-sensitive
proteins.
CYSTEINE OXIDATION
Cysteines are 1 of 2 AA that are naturally incorporated in proteins that contain sulfur, with the other
being methionine. Although oxidation of sulfur on both
AA occurs, the thiol group (-SH) of cysteine is more
reactive than the thioether (-CH2-S-CH3) of methionine. Cysteines are able to participate in a variety of
reactions, in large part because of the ability to exist in many different oxidation states, including thiol,
disulfide, sulfenate, sulfinate, sulfonate, and the thiyl
radical, among others (Figure 1). This flexibility has
allowed cysteines to have a variety of roles, such as
conformation-stability, metal-binding, and catalytic activities.
The major reason for cysteine sensitivity to oxidation
is the ability to generate anionic sulfur at physiological
pH. Most cysteines are not reactive to oxidants because
their microenvironment makes them less nucleophilic or
they are found to be buried within the tertiary or quaternary structure. The typical pKa of a cysteine residue
thiol is approximately 8.5, which is too great to be reactive at physiological pH and must therefore be reduced.
For example, recent results have shown that the reactivity of the functional cysteine (Cys-106) within the
human protein, DJ-1 or Park-7, is due to its acceptance
of a hydrogen bond from a protonated glutamic acid
side chain that reduces the pKa of the thiol group to approximately 5.4 (Witt et al., 2008). In the case of many
cysteine proteases, the formation of the anionic sulfur
is generated by the action of a participating histidine
within the catalytic dyad or triad.
In cases in which the cysteine is buried and inaccessible, they generally remain nonreactive. It should be
noted, however, that oxidation of buried sulfur groups
is possible, but it is both rate and oxidant dependent.
For example, Keck (1996) observed that although the
oxidizing agent t-butyl hydroperoxide was not able to
oxidize buried methionines within interferon-γ, hydrogen peroxide (H2O2) was able to penetrate and oxidize
all 5 methionines within this protein.
CYSTEINE REACTIVE SPECIES
Although the oxidants discussed subsequently are able
to modify other AA, resulting in the modification of enzymatic function, this discussion is focused on those aspects that involve cysteines. Three major subdivisions
of oxidizing species can modify cysteines: reactive oxygen, nitrogen, and sulfur species. It should also be noted that not all oxidants are equivalent, with some being
free radicals (i.e., containing an unpaired electron) and
others being 2-electron oxidants. Each oxidant will also
have different reaction rates with thiols and be differentially reduced by the antioxidant systems, as discussed
below. Oxidants elicit different responses, depending on
where they are generated and with what targets they
are able to react. Within these subdivisions, numerous
types of biologically relevant oxidizing molecules result
Redox regulation of cysteine-dependent enzymes
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Figure 1. Schematic of the major forms of oxidized cysteines observed in biological systems. Peroxide oxidation generates sulfenic acid, which
is an initial step in the formation of other forms of oxidation, including sulfinic and sulfonic acids as well as disulfide bonds. Nitric oxide will oxidize susceptible cysteines, resulting in S-nitrosylation. S-Glutathionylation is a commonly observed modification caused by reaction with oxidized
glutathione. Free radicals that interact with sulfhydryl will generate a reactive sulfur species in the form of a thiyl radical. With the exception of
sulfonic acid, all are reversible, although only peroxiredoxin has been found to be reversible by sulfiredoxins to reduce sulfinic acid. In general,
there is overlap among the antioxidant systems that allows them to reverse several different forms of cysteine oxidation, with a few exceptions in
free radical removal. In some cases, it has also been found that thioredoxin or glutaredoxins are more specific for removal of oxidants from specific
cysteines, which must be determined experimentally. Black ovals represent protein, G represents glutathione, R represents any protein, and GSSG
represents glutathione disulfide.
in cysteine oxidation, including H2O2, superoxide, nitric
oxide (NO), peroxynitrite, and others. These modifications are most often reversible and result in different
cysteine modifications, including disulfide bonds, S-glutathionylation, S-nitrosylation, thiyl radical formation,
and formation of sulfenic, sulfinic, and sulfonic acids.
The most common oxidants and their activities are described briefly below.
Hydrogen peroxide is a nonradical oxidant that is
generated in various compartments throughout the cell
by a variety of sources, such as during the dismutation
of superoxide, as a receptor-signaling molecule, or in
immune-response cells. Peroxide is membrane permeable and is an important physiological redox regulator
with relatively low reactivity, resulting in a long halflife. Peroxide can also react with metals, such as cop-
per or iron, to generate a hydroxyl radical. However,
because the hydroxyl radical is highly reactive, with a
short half-life, and the conditions under which it can be
generated are relatively rare in biological systems, its
significance is unclear (Pacher et al., 2007).
Peroxide oxidation contributes to disulfide bond
formation, as well as to formation of sulfenic, sulfinic, and sulfonic acids. Peroxide-mediated oxidation is
typically reducible, making it an excellent cell-signaling
molecule. Interestingly, until recently it was thought
that both sulfinic and sulfonic acids were nonreducible.
However, Park et al. (2009) showed that enzymes (i.e.,
sulfiredoxins) are present in both yeast and mammals
that are capable of reducing sulfinic acid to sulfenic
acid, at least within the antioxidant enzyme family of
peroxiredoxins.
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Guttmann
−
The superoxide anion (e.g., ∙O2 ) is a radical oxidizing species that is generated by respiration in the
mitochondria and by the immune system. Superoxides
will, either spontaneously or through a catalyzed reaction with superoxide dismutase, decompose to peroxide
(Pacher et al., 2007). Although debatable, it is through
the formation of peroxide and other metabolic products
that superoxide likely exerts its effects on the cell.
Nitric oxide, also known as epithelium-derived relaxation factor, is a vasodilator that is enzymatically
produced by various isoforms of NO synthase, using
arginine as a substrate. Although its main function appears to be as a signaling molecule through the activation of guanylate cyclase, this free radical has emerged
as a significant molecule in the study of redox regulation through multiple actions (Pacher et al., 2007).
Although NO can be converted to higher orders of reactive nitrogen species, the 2 major reactive forms found
in cells are those of S-nitrosylation and peroxynitrite.
S-Nitrosylation is the covalent addition of an NO group
to critical anionic sulfur within cysteine to form an Snitrosothiol derivative. Nitric oxide also reacts with superoxide to form the more reactive peroxynitrite, which
has a half-life sufficient to cross biological membranes
(Pacher et al., 2007). Peroxynitrite can oxidize thiols
directly, resulting in the generation of a sulfenic acid
that may subsequently form a disulfide. Alternatively,
the decomposition products of peroxynitrite, the hydroxyl radical and nitrogen dioxide (NO2), will result
in the generation of a thiyl radical. Although cysteines
are a significant target of peroxynitrite, other AA, such
as tyrosine, methionine, tryptophan, and histidine, are
modified. As a biological oxidant, however, peroxynitrite is a significant factor in the regulation of cysteinedependent enzymes.
ANTIOXIDANT SYSTEMS
For a protein to be reduced, the reducing agent or
antioxidant must be oxidized during the reaction. Thus,
the general goal of a cell, to remove oxidants, will be to
shuttle the electron(s) from the oxidized species to generate water or oxygen. This reduction reaction chain is
a multistep process utilizing several cofactors, including
flavin adenine dinucleotide (FAD), NADH, or reduced
NAD phosphate (NADPH). Without a final electron
acceptor with the ability to eventually remove the electron pair (note the difference between 2-electron nonradical oxidants and 1-electron free radicals), there
would be no net change in redox state within the cell.
The cell has an array of defensive mechanisms in its
arsenal that deal with the formation of oxidized species. These include enzymes [e.g., superoxide dismutase
(SOD) and catalase], in addition to nonenzymatic
pathways, such as cysteine, glutathione, and vitamins
(e.g., vitamins C and E). In addition to those factors
that are most directly involved in the reduction of oxidized species, numerous cofactors, such as FADH/H2,
NADH, and NADPH, must also be maintained.
There are 2 main forms of SOD in mammalian cells,
MnSOD (SOD2; localized to mitochondria) and CuZnSOD, with 2 types of CuZnSOD that are found either
intracellularly (SOD1) or extracellularly (SOD3). These
enzymes are responsible for the conversion of superoxide
to H2O2, although this occurs slowly and spontaneously
as well. Although superoxide is an oxidizing radical,
its direct action is limited, and it would seem that the
main reason for maintaining increased SOD activity is
to prevent the formation of peroxynitrite, the reaction
between superoxide and NO (Pacher et al., 2007).
Catalase and peroxidases are enzymes that remove
H2O2. Hydrogen peroxide is a product of various oxidases, such as those found in peroxisomes or in the endoplasmic reticulum, and the dismutation of superoxide. Removal of H2O2 is important because peroxide is
converted, through the Fenton reaction (i.e., the reaction of peroxide with ferrous iron), into a hydroxyl radical that is a highly reactive but short-lived oxidant.
Thioredoxin (TRx) is a ubiquitous small protein, approximately 12 kDa in size, consisting of 105 AA with
a conserved Cyx-Xaa-Xaa-Cys motif in its active site,
that reduces target proteins by exchanging disulfide to
dithiol. Its reducing activity is maintained by NADPH
and TRx reductase (TRxR; Ren et al., 1993). Mammalian cells contain 2 TRx systems, the cytosolic TRx
and TRx reductase (TRxR1) and mitochondrial TRx2TRxR2 (Damdimopoulos et al., 2002). In mammals the
TRxR exist as homodimeric selenoenzymes containing
an FAD and a C-terminal selenocysteine residue in the
active site. Thioredoxin reductase 1 is cytosolic but can
translocate to the nucleus or be exported from the cell
even though it lacks a targeting sequence. Targets of
TRx include hypoxia-inducible factor 1, nuclear factorκB, activator protein 1, and the tumor suppressor p53.
In addition, TRx have been shown to scavenge hydroxyl radicals.
Glutaredoxins (GRx) are also small proteins of 10
to 16 kDa in size that have 2 classes: the dithiol GRx,
which contain CxxC, and the more recently discovered
monothiol, which lack the C-terminal cysteine (Lillig et
al., 2008). The GRx system is composed of glutathione,
glutathione reductase, NADPH, and GRx. There are
several GRx isoforms that do not completely overlap
in activities with each other or with the TRx, although
many are shared. Some localization differences exist
because GRx1 is localized mainly in the cytosol and
the intermembrane space of the mitochondria, whereas
GRx2 localizes to either the matrix of the mitochondria
or the nucleus (Pai et al., 2007). Glutaredoxin 2 can
also be reduced by TRxR, which is unique among the
GRx. Glutaredoxin 5, a monothiol GRx, is found in the
mitochondria that are required for the activity of ironsulfur enzymes.
Glutathione peroxidases (GPx) are selenocysteinecontaining proteins primarily involved in the reduction
of H2O2 and lipid peroxides (Lu and Holmgren, 2009).
Several isoforms (GPx1 through GPx6) exist, with at
least one mammalian isoform that does not utilize sele-
Redox regulation of cysteine-dependent enzymes
nocysteine. Similar to the other enzymatic systems,
these enzymes are found throughout the cell with cytosolic, nuclear, mitochondrial, and secreted forms. Some
are tissue specific, such as GPx5, which plays a role in
protecting DNA in sperm from oxidative damage, and
GPx6, which is localized to the olfactory system.
Peroxiredoxins (Prx) are ubiquitously expressed and
catalyze the removal of H2O2 and lipid hydroperoxides
as well as peroxynitrite. At least 6 Prx are found in
mammalian cells and can be divided into 3 subclasses:
1) typical 2-Cys-Prxs (Prx I through Prx IV homodi­
mers); 2) atypical 2-Cys-Prx (Prx V); and 3) 1-Cys-Prxs
(Prx VI). For the typical Prx, the active-site cysteine
is oxidized to sulfenic acid, which then reacts to form
an intermoleculer disulfide, which is reduced by TRx.
It is possible for the sulfenic acid to be further oxidized
to sulfinic acid, which can be reduced by sulfiredoxin
(Park et al., 2009). It has also been found that ascorbate may be used by some Prx to regenerate the active
site thiol (Monteiro et al., 2007).
Glutathione (GSH) is a tripeptide (γ-glutamylcysteinyl-glycine) that, along with glutathione disulfide
(GSSG), plays a significant role in antioxidant defense.
This redox couple (GSH:GSSG) is a major determinant
of the redox state inside the cell, where they are found
at millimolar concentrations (Jones, 2008). In addition
to being able to regenerate various antioxidant systems
and serve directly as an antioxidant, GSH also acts as
a source for the AA cysteine. Once oxidized, GSSG
is reduced in a NADPH-dependent process by GSH
reductase. Lack of NADPH leads to increased GSSG
abundance, which is important in red blood cells that
lack mitochondria and that therefore rely on the glucose-6-phosphate dehydrogenase pathway. A result of
insufficient glucose-6-phosphate dehydrogenase activity is hemolytic anemia, which is observed in millions
worldwide and can be caused by certain drugs, such as
antimalarial compounds (Aliciguzel and Aslan, 2004).
Cysteine is the major extracellular antioxidant (Moriarty-Craige and Jones, 2004). Once oxidized, it forms
the low molecular weight disulfide, CySS, or other
forms of oxidized thiols, as described previously. However, it is primarily found as CySS because the extracellular environment is relatively oxidizing. Although
this pool is integrated with the other major intracellular antioxidant, GSH, the 2 are not in equilibrium (Go
and Jones, 2008); however, they influence each other
because GSH formation is dependent on the presence
of cysteine and vice versa. Although cysteine is not considered an essential AA, it is derived from methionine,
which is essential. Because very young or very old animals may have difficulty absorbing sufficient quantities
from the diet to produce adequate concentrations of
cysteine, sufficient methionine and cysteine in the diet
is vital (Rutherfurd and Moughan, 2008).
Vitamin C (ascorbate) is an essential nutrient in humans, but not in most animals. It serves as a watersoluble antioxidant and cofactor for the regeneration
of some enzymatically important thiols (Mandl et al.,
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2009). As an antioxidant, ascorbate is typically involved
in nonenzymatic 1-electron (i.e., radical) transfers and
is therefore important in the removal of superoxide, hydroxyl, and peroxyl radicals. The monodehydroascorbate produced by the scavenging of radicals is ultimately reconverted to ascorbate by enzymatic conversions
that use NADH, NADPH, or GSH. In the presence of
heavy metals, ascorbate can become pro-oxidant, although the frequency of this occurrence in biological
systems is unclear (Mandl et al., 2009).
Vitamin E refers to a group of 8 related compounds,
among which α-tocopherol shows the greatest biological activity. It is a lipid-soluble antioxidant whose main
role is the prevention of membrane lipid peroxidation.
Once oxidized, vitamin E is likely regenerated by either
ascorbate or β-carotene.
EFFECTS OF REDOX
ON CELLULAR FUNCTION
The oxidants and antioxidants work as a complete
system, which has implications for the physiological
regulation and pathological impact of numerous proteins. The following section highlights, using several examples, the manner in which oxidative stress contributes to various cellular pathways of major significance
to all researchers interested in redox biology.
Redox Regulation of Transcription
Heat shock transcription factor 1 (HSF1), an important regulator of inducible heat shock proteins (Hsp)
in mammals, is one example of a transcription factor modulated by redox state. In its monomeric form,
HSF1 is located in the cytosolic fraction. When cells
are placed under stress, HSF1 forms a homotrimer that
is translocated to the nucleus, where it binds to a heat
shock element within DNA and activates transcription
of Hsp, such as Hsp70 and Hsp90. Lu et al. (2008)
recently observed that disulfide bridges control the formation of the homotrimer in vitro. They found that the
2 intermolecular disulfide bonds had differential sensitivities to redox state, which they manipulated by the
addition of different concentrations of the thiol-specific
reducing agent dithiothreitol. In the presence of stress
and oxidizing conditions, an intramolecular disulfide
bond predominated, which prevented activation of
HSF1. Under stress and mildly reducing conditions, the
intramolecular bond was broken and HSF1 could not
form the required trimer. However, under more highly
reducing conditions, the homotrimer disulfide bridges
were broken. This example illustrates the fine sensitivity of the redox state that, in this case, controlled protein function through disulfide bond formation.
Nuclear factor E2-related factor 2 (Nrf2) is a ubiquitously expressed master transcription factor that
regulates antioxidant response elements, which in turn
mediate expression of antioxidant proteins. Kelch-like
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ECH-associated protein (Keap1) is a cysteine-rich,
zinc-binding protein that is a negative regulator of
Nrf2. After many years of study, a unique mechanism
was found in which Keap1 targets Nrf2 for rapid turnover by proteolytic degradation by the proteasome
(Sekhar et al., 2002). As a major factor in the inducible
upregulation of antioxidant enzymes, the Nrf2-Keap1
interaction is responsive to changes in the redox state,
which are now believed to be mediated not by Nrf2 itself, but rather by a mechanism associated with Keap1.
Although there is some debate over the precise mechanism and elements involved in the redox regulation in
vivo, it is clear that modifications of one or more of
the key cysteines within Keap1 are involved and likely
result in the dissociation of Keap1 from Nrf2 (Piccirillo
et al., 2009).
Examples of Redox-Regulated Enzymes
Although several of the key antioxidant systems are
cysteine-dependent enzymes, this section focuses on a
select group of thiol-dependent enzymes that are not
directly related to maintaining the redox state. Evidence for their oxidation is presented first, followed by
an integrated discussion of the impact of their oxidation on cellular function to illustrate the importance of
recognizing the influence of oxidation in data interpretation and experimental design considerations.
Protein tyrosine kinases (PTK) are a family of kinases that contain a conserved active-site cysteine
(His-Cys-X-X-Gly-X-X-Arg-Ser/Thr). Because of its
microenvironment, this active-site cysteine has a low
pKa, which makes it susceptible to redox regulation by
forming the highly sensitive thiolate anion at physiological pH. A reduced cysteine within the active site
acts as a nucleophile, which is required to phosphorylate PTK substrates. Oxidation of cysteine results in a
less nucleophilic center, which cannot participate in the
reaction. Protein tyrosine phosphatase 1b is a classical example of such PTK redox regulation. Protein tyrosine phosphatase 1b has been found to be reversibly
oxidized by several oxidizing agents, notably H2O2, to
form sulfenic acid (Denu and Tanner, 1998), as well as
by glutathionylation (Barrett et al., 1999a,b). The net
result of oxidative stress in this case is an increase in
tyrosine phosphorylation that occurs under physiological conditions, such as during insulin signaling through
the insulin receptor.
Creatine kinase is an important enzyme in energy
transfer because it catalyzes the transfer of high-energy
phosphate from ATP to creatine. Multiple isoforms exist that are tissue specific, as well as being found in both
the cytosol and mitochondria. Creatine kinase isoforms
contain up to 4 cysteines, of which only one appears to
be necessary and sufficient for full creatine kinase activity and is the only one that appears to be physiologically relevant (Konorev et al., 2000; Liu et al., 2008).
Modifications to creatine kinase include modification
by reactive nitrogen species, disulfide formation, and
S-glutathionylation (Konorev et al., 2000; Reddy et al.,
2000; Liu et al., 2008).
Calpains are cysteine-dependent proteases that are
involved in several physiological pathways and are
thought to contribute to a variety of pathological states
(Goll et al., 2003). They have a catalytic triad, related
to papain, consisting of a cysteine, histidine, and asparagine. Calpains exist as multiple isoforms with ubiquitous and tissue-specific isoforms that are primarily
found in various muscle types, which has led to their
study in meat tenderization, as discussed below. Similar to other thiol-dependent enzymes, the active-site
cysteine generates an anionic sulfur group during catalysis that is subject to oxidative attack. The various
members of this family of enzymes have been found
to be inhibited by a variety of oxidants, most notably
peroxide and NO, resulting in reversible enzyme inactivation (Guttmann et al., 1997; Guttmann and Johnson,
1998; Koh and Tidball, 2000).
The importance of recognizing the groups discussed
above is that they represent the typical types of oxidative modifications that are observed and the different
outcomes that result. It should be easily appreciated
that changes in redox balance can have both focused
and broad effects, even though only a single cysteine
may be modified within a given protein. For example,
in cases in which redox state is used as a primary modulator of enzyme activity, such as with protein tyrosine
phosphatase 1b and its regulation by insulin, a change
in redox has a controlled and relatively localized influence. However, when more systematic changes in redox
occur, the changes can be cell-wide, resulting in modifications of many thiol-containing proteins because of
transcription-mediated responses. The sensitivity and
importance of the AA cysteine should also be evident
because in each case, only 1 or 2 cysteines within a
quarternary structure that can be targeted by oxidative
stress will have a significant influence on the function
of a protein. That is, not all cysteines that are oxidized
within a given protein will contribute to any change in
activity. In addition, it should be clear that virtually
all the types of oxidation that are chemically possible
are observed. However, the possible oxidations observed
in vitro may not have physiological meaning, and it is
therefore important to have a good understanding of
the redox compartment in which a protein under study
is likely to be found and to verify any in vitro studies
with in vivo experimentation.
REDOX-RELEVANT ISSUES
TO ANIMAL SCIENTISTS
The previous examples can illustrate several broad
areas for the animal scientist to consider. These include
understanding the effects of 1) experimental conditions
on results that may be related to oxidative stress, 2)
nutrition on redox-regulated biological pathways, 3)
oxidative stress on aged animals, and 4) postmortem
oxidation of food.
Redox regulation of cysteine-dependent enzymes
Experimental Considerations
A common reagent added to reactions is a thiolreducing agent, usually dithiothreitol or the related
β-mercaptoethanol. Often it is added to prepare samples for analysis, such as with gel electrophoresis, and
is needed to fully unfold proteins by removing disulfide
bridges. However, when added to cell or tissue homog­
enates that will be used for bioassays, this also results
in adjusting the redox state from what it was at the
time the samples were taken. In many instances, this
may not have a significant impact on the results or
data interpretation. However, if there is an active thiol
group within the protein of interest or a regulator of
this protein (e.g., as observed with the Keap1-Nrf2dithiothreitol experiments described previously), then
the addition of increased concentrations of reducing
agents may lead to an over- or underestimation of activity. Recently, we demonstrated this phenomenon in
brain homogenates, showing that the estimation of calpain activity was clearly affected by the presence oxidation related to the disease (Marcum et al., 2005). This
conclusion was reached by examining calpain activity
in homogenates that were treated with or without dithiothreitol. We found that although earlier estimations of calpain activity appeared elevated in disease
vs. control states, this was likely due to the addition
of dithiothreitol to the assay, which negated any influence of the oxidative stress known to be present in
the disease state. This approach itself adds a layer of
complexity because the homogenization or other manipulations that must be taken to prepare samples will
change the redox state. However, with proper controls,
an examination of samples under normoxic and reducing conditions would yield useful results with respect to
the potential impact of oxidative stress.
A second consideration is the use of specific antioxidant systems as measures of oxidative stress. As
indicated previously, these systems are in some ways
interdependent because they share some common pathways or cofactors or because they exist within the same
compartment. However, an indication of the redox level
of one system does not necessarily translate into an accurate picture within each compartment. As reviewed
by Jones (2008), the GSH:GSSG is only one measure of
the redox state of the cell, so care must be taken when
attempting to extrapolate changes in the levels or ratio
of this redox couple. As in the examples above, several
enzymatic processes play vital roles in the reduction
of oxidized thiols, and although they are regenerated
in large part by the glutathione system (directly or indirectly), a decrease in reduced glutathione does not
necessarily significantly modulate the capacity of other
redox compartments. Additionally, alterations in the
GSH:GSSG redox couple would not necessarily indicate
the redox state of a protein of interest. Fortunately,
with the increased availability of highly sensitive thioldetecting reagents and equipment, it is now possible
1303
to monitor specific cysteines to determine the extent
to which they are oxidized or reduced. Thus, an understanding of the global redox state with GSH:GSSG
or Cys:CySS measures is valuable, but care should be
taken when interpreting these values in the context of
a particular activity of interest.
A third area to be evaluated with respect to the role
of oxidative stress in experimentation is the influence
of drug treatments that may perturb the redox balance.
Of particular note are compounds that alter mitochondrial energetics. As an important source of superoxide,
mitochondrial efficiency can have a significant impact
on the redox state of the inner and outer mitochondrial
spaces, as well as on the entire cell. As one example,
doxorubicin, a chemical used in the treatment of solid
tumors, is restricted in its use in part because of its
cardiotoxicity. There is strong evidence that toxicity
is caused by the interaction of doxorubicin with mitochondrial proteins, resulting in increased production of
superoxide. One example of this has been observed with
creatine kinase. It was found that in mitochondria isolated from animals treated with doxorubicin, creatine
kinase was S-glutathionylated, which was reversible by
overexpression of glutaredoxin (Diotte et al., 2009).
These results clearly show that care must be taken to
consider the impact of therapeutics that may have unexpected outcomes on redox-regulated enzymes.
The influence of oxidants and reductants, including
dithiothreitol, on electrophoresis and fluorescent-based
assays also needs to be considered. Gel electrophoresis
is a common analytical approach, and we have found
that while conducting in vitro experiments with increased concentrations of dithiothreitol, this and other
reductants and oxidants alter the ability of proteins to
be visualized by Western blotting (R. P. Guttmann,
unpublished data). Although we have not attempted
a systematic study of this phenomenon, we have found
that the more reducing agent is used, the stronger will
be the signal observed by Western blot. To overcome
this potential limitation, the use of freshly prepared
and equal concentrations of reducing agents added to
the final SDS-sample buffer used in preparation for the
loading of protein is mandatory and has been successful
in preventing this artifact. In attempting to determine
the extent to which oxidants or reductants affect activities, highly sensitive fluorescent-based activity assays
have become more common. However, using such assays under highly oxidizing or reducing conditions must
be done with caution. We have found that reductants
can have direct effects on the fluorescence of coumarinbased moieties, although we have not evaluated other
types of fluorescent probes. To address this issue, it
is imperative that testing of all control and treatment
conditions be done in the presence or absence of the
enzyme or molecule under study to evaluate the extent
to which the oxidant or reductant may be affecting the
fluorescent signal. Alternatively, measurements of the
fluorescent moiety in the buffers and conditions being
1304
Guttmann
tested can be taken to confirm that its fluorescence is
not being directly affected by the presence of increased
concentrations of oxidizing or reducing agents.
Nutrition
The impact of proper nutrition on the redox state
of the intracellular and extracellular environment is
another important factor to be considered by animal
scientists. First, there must be an awareness of the
metabolic pathways utilized by a particular species.
As noted previously, bioavailability of methionine and
cysteine is critical to maintaining a proper physiological
environment and adequate concentrations of cysteine.
In addition, knowledge of the proper supplementation
of vitamins, such as ascorbate, is vital because not all
animals have the same biochemical pathways available
to process nutrients in feed, including minerals and vitamins. Selenium is another potential supplement for
which abundance should be evaluated because of the
importance of selenium in redox biology. Because increasing the quantity of antioxidants has general positive outcomes, the use of natural or synthetic compounds to support the natural antioxidant systems is
a tantalizing approach. One well-studied example is Nacetylcysteine and its related compounds. These compounds have been tested in both animals and humans
and appear to be well tolerated and effective at decreasing oxidant-mediated damage (Ferreira and Reid, 2008;
Banerjee et al., 2009; Ciftci et al., 2009; Lu et al., 2009;
Tunc et al., 2009). Whether these positive outcomes are
caused by providing an additional source of cysteine
or whether N-acetylcysteine is acting directly as an
antioxidant is still under investigation. However, this
is strong evidence that N-acetylcysteine or other compounds that have potent antioxidant activity are good
candidates for study in the prevention or treatment of
oxidative stress. In addition to N-acetylcysteine, other
compounds, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Mitchell et al., 1991), are available and
being developed that seek to improve health by decreasing oxidative stress.
Studies of the importance of redox state on the thiol
groups in seeds have also been under investigation. In a
study to evaluate the role of disulfides in the assembly
of 11S storage globulins, it was found that manipulation of the redox state in vitro using various concentrations and ratios of GSH:GSSG resulted in an increase
in the rate of 11S trimer assembly and hexamer formation (Jung et al., 1997). Proper regulation of thiols in seeds was also studied in experiments showing
the importance of regulating the formation of disulfide
bonds during germination. In that study, the authors
concluded that disulfide bonds of seed storage proteins
were required to be reduced, likely by TRx, to be degraded and mobilized during germination. More recent
work has extended these findings to more fully evaluate
the contribution of TRx in seed germination, in addition to discovery of the role of a cysteine-protease in
the processing of the embryo-specific protein 2 (Yano
et al., 2001).
Aging Animals
It is clear that oxidative stress is closely linked with
the aging process. Although it remains to be determined whether it is cause or effect, results have consistently shown that as an animal ages, markers of oxidation increase. There is good evidence that increases in
oxidative stress with age are detrimental, although the
extent to which increasing antioxidant concentrations
prolong life span remains hotly debated. Much of the
work that laid the foundation for evaluating oxidative
stress and life span has naturally been done in lower order organisms. Nevertheless, these initial observations,
along with animal and human data, demonstrate the
potential power of redox regulation in improving the
quantity and quality of life. Although still under intense
investigation, other work has shown that caloric restriction results in decreased oxidative stress and has been
proposed as one mechanism to explain the increase in
longevity associated with a restricted diet (Sohal and
Weindruch, 1996; Lass et al., 1998; Zainal et al., 2000;
Martin et al., 2007). As more information has been accumulating about the importance of proper nutrition,
it would seem clear that proper nutrition related to
maintaining sufficient raw materials (such as cysteine,
methionine, selenium, and vitamins C and E) to allow
for optimal antioxidant synthesis is an important area
for animal scientists. It would be expected that improving the redox balance in newborn and young animals
would positively affect the overall health of animals. In
addition, maintaining redox balance could have more
profound influences on aged-related changes, such as
fertility, muscle fatigue, stamina, and other important
characteristics that are desirable.
Postmortem Changes and the Role
of Oxidative Stress
Postmortem changes affect many forms of research
by creating artifacts and are thus a constant area of
concern. However, in the case of food sciences, understanding the impact of postmortem changes is vital.
As discussed previously, calpains were observed to be
sensitive to a variety of oxidants, resulting in enzyme
inactivation. Because several calpain substrates are cytoskeletal components, as well as because of the increased muscle concentration of calpains, evaluation
of the postmortem activity of calpain was undertaken.
It was found that postmortem calpain activity was
a significant factor contributing to meat tenderness
(Kretchmar et al., 1990; Koohmaraie, 1992; Wheeler et
al., 1992; Whipple and Koohmaraie, 1992). More recent
studies have found that meat tenderness is decreased
by oxidative inactivation of calpains and that treatment with antioxidants can increase meat tenderness,
Redox regulation of cysteine-dependent enzymes
which is at least partially attributable to preventing
oxidative inactivation of calpains (Rowe et al., 2004).
Prevention of oxidative stress postmortem is also important in packaging and storage because the impact of
oxidative stress makes food less appealing, less palatable, and, from a food safety standpoint, potentially
dangerous. The understanding of oxidation prevention
in the market is observed by noting the use of techniques that prevent oxidation to store and package
foods for human consumption. In addition to inhibiting
the growth of bacteria and other pathogens, decreasing
oxidation of the food product will prevent cysteine oxidation, which could contribute to protein aggregation
through disulfide bonds. It would seem that a balance
is required because high-thiol-dependent enzymatic activities postmortem could also be a factor contributing
to food spoilage through the actions of cysteine-dependent proteases (Chen et al., 2008).
Conclusions
In summary, this review has attempted to provide a
sense of the scope of the potential roles of thiol oxidation in cellular function and has instructed the reader
on the importance of considering the influence of redox potential in experimentation with animals, animal
products, and food. The reader is also directed to some
of the outstanding reviews of the details of each oxidant
and antioxidant system, which contain more specific
information regarding the chemistry of the redox reactions and enzymatic pathways related to cysteine oxidation (Giles et al., 2003; Moriarty-Craige and Jones,
2004; Pacher et al., 2007; Go and Jones, 2008; Jones,
2008; Winterbourn and Hampton, 2008; Brandes et al.,
2009).
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