From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
The demonstration that oxidatively modified forms
of proteins accumulate during aging, oxidative stress, and in some
pathological conditions has focused attention on physiological and
non-physiological mechanisms for the generation of reactive oxygen
species (ROS)1 and on the
modification of biological molecules by various kinds of ROS. Basic
principles that govern the oxidation of proteins by ROS were
established in the pioneering studies of Swallow (1), Garrison (2, 3),
and Scheussler and Schilling (4) who characterized reaction products
formed when proteins were exposed to ionizing radiation under
conditions where only ·OH, O Oxidation of the Protein Backbone As is illustrated in Fig. 1,
oxidative attack of the polypeptide backbone is initiated by the
·OH-dependent abstraction of the
The generation of alkoxyl radicals (Fig. 1, Reactions h
and g) sets the stage for cleavage of the peptide
bond by either the diamide or
Peptide bond cleavage can occur also as a result of ROS attack of
glutamyl, aspartyl, and prolyl side chains. As described by Garrison
(2), ·OH-dependent abstraction of a hydrogen atom
from the Fig. R3.
Based on the observation that the number of peptides formed during
radiolysis of proteins is approximately equal to the number of prolyl
residues, Schuessler and Schilling (4) proposed that oxidation of
prolyl residues would lead to peptide bond cleavage. This was verified
by studies of Uchida et al. (5) showing that oxidation of
proline residues leads to the formation of 2-pyrrolidone and
concomitant peptide bond cleavage (Reaction
R4). Because acid hydrolysis of
2-pyrrolidone yields 4-aminobutyric acid, the presence of
4-aminobutyric acid in protein hydrolysates is presumptive evidence for
peptide bond cleavage by the proline oxidation pathway.
Fig. R4.
Oxidation of Amino Acid Side Chains All amino acid residues of proteins are susceptible to oxidation
by ·OH. However, the products formed in the oxidation of some
residues have not been fully characterized. Table
I lists some of the products formed
during the oxidation of the residues that are most susceptible to
oxidation.
Table I.
Amino acids most susceptible to oxidation
2, or a mixture of both was
made available. Results of these studies demonstrated that the
modification of proteins is initiated mainly by reactions with
·OH; however, the course of the oxidation process is determined by the availability of O2 and O
2 or its protonated
form (HO·2). Collectively, these ROS can
lead to oxidation of amino acid residue side chains, formation of
protein-protein cross-linkages, and oxidation of the protein backbone
resulting in protein fragmentation. In the meantime, it has been shown
that other forms of ROS may yield similar products and that transition
metal ions can substitute for ·OH and O
2 in some of the
reactions.2
-hydrogen atom
of an amino acid residue to form a carbon-centered radical (Fig. 1,
Reaction c). The ·OH needed for this reaction may be
obtained by radiolysis of water or by metal-catalyzed cleavage of
H2O2 (Reactions a and b).
The carbon-centered radical thus formed reacts rapidly with
O2 to form an alkylperoxyl radical intermediate (Reaction
d), which can give rise to the alkylperoxide (Reaction
f), followed by formation of an alkoxyl radical (Reaction
h), which may be converted to a hydroxyl protein derivative
(Reaction j). Significantly, many of the steps in this
pathway that are mediated by interactions with
HO·2 can be catalyzed also by
Fe2+ (Reactions e, g, and
i)2 or by Cu+ (not shown). The
alkyl, alkylperoxyl, and alkoxyl radical intermediates in this pathway
may undergo side reactions with other amino acid residues in the same
or a different protein molecule to generate a new carbon-centered
radical (Reaction 1) capable of undergoing reactions similar to those
illustrated in Fig. 1.
Moreover, in the absence of oxygen, when Reaction
d in Fig. 1 is prevented, the carbon-centered radical may
react with another carbon-centered radical to form a protein-protein
cross-linked derivative (Reaction 2).
Fig. 1.
Oxygen free radical-mediated oxidation of
proteins.
[View Larger Version of this Image (18K GIF file)]
-amidation pathways. Upon cleavage by
the diamide pathway (Fig. 2, Pathway
a), the peptide fragment derived from the N-terminal portion
of the protein possesses a diamide structure at the C-terminal end,
whereas the peptide derived from the C-terminal portion of the protein
possesses an isocyanate structure at the N-terminal end. In contrast,
upon cleavage by the
-amidation pathway (Fig. 2, Pathway
b), the peptide fragment obtained from the N-terminal
portion of the protein possesses an amide group at the C-terminal end,
whereas the N-terminal amino acid residue of the fragment derived from
the C-terminal portion of the protein exists as an
N-
-ketoacyl derivative. Upon acid hydrolysis, the peptide
fragments obtained by the diamide pathway will yield CO2, NH3, and a free carboxylic acid, whereas hydrolysis of the
fragment obtained by the
-amidation pathway yields NH3
and a free
-ketocarboxylic acid.
Fig. 2.
Peptide bond cleavage by the (a)
diamide and (b) -amidation pathways.
[View Larger Version of this Image (10K GIF file)]
-carbon atom of a glutamyl residue, followed by reactions
analogous to Reactions d, f, and h in Fig. 1,
will lead eventually to peptide bond cleavage by a mechanism in which
oxalic acid is formed and the N-terminal amino acid of the peptide
derived from the C-terminal portion of the protein will exist as an
N-pyruvyl derivative (Reaction R3).
Amino acids
Oxidation products
Cysteine
Disulfides, cysteic acid
Methionine
Methionine sulfoxide, methionine sulfone
Tryptophan
2-, 4-, 5-, 6-, and 7-Hydroxytryptophan,
nitrotryptophan, kynurenine, 3-hydroxykynurinine, formylkynurinine
Phenylalanine
2,3-Dihydroxyphenylalanine, 2-, 3-, and
4-hydroxyphenylalanine
Tyrosine
3,4-Dihydroxyphenylalanine,
tyrosine-tyrosine cross-linkages, Tyr-O-Tyr, cross-linked
nitrotyrosine
Histidine
2-Oxohistidine, asparagine, aspartic
acid
Arginine
Glutamic semialdehyde
Lysine
-Aminoadipic semialdehyde
Proline
2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic
acid, glutamic semialdehyde
Threonine
2-Amino-3-ketobutyric acid
Glutamyl
Oxalic acid, pyruvic acid
Cysteine and methionine residues are particularly sensitive to oxidation by almost all forms of ROS. Under even mild conditions cysteine residues are converted to disulfides and methionine residues are converted to methionine sulfoxide (MeSOX) residues. Most biological systems contain disulfide reductases and MeSOX reductases that can convert the oxidized forms of cysteine and methionine residues back to their unmodified forms. These are the only oxidative modifications of proteins that can be repaired. Based on the observation that preferential oxidation of several exposed methionine residues in some proteins has little effect on their biological function, it was proposed that the cyclic oxidation-reduction of methionine residues serves as a "built-in" ROS scavenger system to protect such proteins from more extensive irreversible oxidative modifications (6). This proposition is supported by results of recent studies showing that a "knock-out" strain of yeast lacking MeSOX reductase is more sensitive to H2O2 toxicity than the wild-type strain and that, when grown in the presence of H2O2, the protein and free amino acid pool of the mutant strain contain higher levels of MeSOX than are present in the wild type strain.3
Aromatic Amino Acid ResiduesAromatic amino acid residues are among the preferred targets for ROS attack. As shown in Table I, tryptophan residues are readily oxidized to formylkynurenine and kynurenine and to various hydroxy derivatives; phenylalanine and tyrosine residues yield a number of hydroxy derivatives; histidine residues are converted to 2-oxohistidine, asparagine, and aspartic acid residues.
Reactions with PeroxynitriteWith the discovery that nitric
oxide is a normal product of arginine metabolism and that it reacts
rapidly with O2 to form peroxynitrite (Reaction 5), the
biological effects of peroxynitrite (PN) have been extensively
studied.
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The ability of PN to nitrate tyrosine residues and oxidize methionine
residues of proteins is dependent upon the availability of
CO2. In the absence of CO2, PN is in
equilibrium with an activated form (PN*) of unknown structure that
reacts rapidly with methionine residues to form MeSOX (12), but in the
presence of CO2, PN is almost instantly converted to a
derivative (possibly O=NOOCO2 or
O2NOCO2
) that can nitrate aromatic
compounds (13-16). Accordingly, the nitration of tyrosine and the
oxidation of methionine residues of proteins are mutually exclusive
processes that are differentially regulated by the availability of
CO2, as illustrated in Scheme 1.
Scheme 1.
Curiously, in the case of GS, there is little or no nitration of tyrosine residues in the complete absence of CO2, and no oxidation of methionine residues occurs in the presence of physiological concentrations of CO2 (i.e. 5% CO2, pH 7.4). Even so, the PN-dependent oxidation of methionine residues in the absence of CO2 and the nitration of tyrosine residues in the presence of CO2 both convert GS to a form with regulatory properties similar to those obtained by enzyme-catalyzed adenylylation of a single tyrosine in each subunit of the enzyme (17).
Generation of Protein Carbonyl Derivatives
As already noted, oxidative cleavage of proteins by either the
-amidation pathway (Fig. 2) or by oxidation of glutamyl side chains
(Reaction R3) leads to formation of a peptide in which the N-terminal
amino acid is blocked by an
-ketoacyl derivative. However, as shown
in Table I, direct oxidation of lysine, arginine, proline, and
threonine residues may also yield carbonyl derivatives. In addition,
carbonyl groups may be introduced into proteins by reactions with
aldehydes (4-hydroxy-2-nonenal, malondialdehyde) produced during lipid
peroxidation (Fig. 3A)
(18-20) or with reactive carbonyl derivatives (ketoamines,
ketoaldehydes, deoxyosones) generated as a consequence of the reaction
of reducing sugars or their oxidation products with lysine residues of
proteins (21-23) (glycation and glycoxidation reactions) (Fig.
3B). The presence of carbonyl groups in proteins has
therefore been used as a marker of ROS-mediated protein oxidation, and
several sensitive methods for the detection and quantitation of protein
carbonyl groups have been developed (24). As judged by the presence of
carbonyl groups, it has been established that protein oxidation is
associated with aging, oxidative stress, and a number of diseases.
Oxidative Stress-induced Protein Oxidation
Elevated levels of oxidized protein are present in animals and cell cultures following their exposure to various conditions of oxidative stress. Thus, exposure of animals or cell cultures to either hyperoxia, forced exercise, ischemia-reperfusion, rapid correction of hyponatremia, paraquat toxicity, magnesium deficiency, ozone, neutrophil activation, cigarette smoking, x-radiation, chronic alcohol treatment, or mixed function oxidation systems leads to an increase in the level of oxidized protein.4
Protein Oxidation and AgingAging is associated with the accumulation of inactive or less active, more heat-labile forms of numerous enzymes (25, 26). The possibility that these age-related changes are due, at least in part, to oxidative modification is indicated by the facts. (a) In vitro exposure of enzymes to ROS elicits changes in catalytic activity, heat stability, and proteolytic susceptibility similar to those that occur during aging (27-30). (b) Brief exposure of animals to oxidative stress leads to alterations in enzymes similar to that associated with aging (31, 32). (c) Old animals are more susceptible than young animals to protein damage during oxidative stress, e.g. x-radiation, H2O2 (33, 34). (d) There is an age-related increase in the carbonyl content of protein in human brain (35), gerbil brain (36), eye lens (37), rat hepatocytes (32), whole body protein of flies (38), and human red blood cells (28). (e) The carbonyl content of protein in cultured human fibroblasts increases exponentially as a function of the age of the fibroblast donor (28). (f) There is an inverse relationship between regimens that lead to an increase in life span and regimens that lead to an increase in protein carbonyl content and vice versa (39). For example, diet (caloric) restriction of rats (39) and mice (40) leads to an increase in life span and to a decrease in the level of protein carbonyls. When compared at the same chronological age, strains of short lived houseflies contain higher levels of oxidized proteins than their longer lived cohorts (41).
Protein Oxidation and DiseaseAccumulation of oxidized protein (protein carbonyls) is associated with a number of diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, respiratory distress syndrome, muscular dystrophy, cataractogenesis, rheumatoid arthritis, progeria, and Werner's syndrome.4 Although the level of carbonyl has not been directly determined, there is reason to believe that oxidative modification of proteins is implicated also in atherosclerosis, diabetes, Parkinson's disease, essential hypertension, cystic fibrosis, and ulcerative colitis.4
Accumulation of Oxidized Protein
The intracellular level of oxidized protein reflects the balance
between the rate of protein oxidation and the rate of oxidized protein
degradation. This balance is a complex function of numerous factors
that lead to the generation of ROS, on the one hand, and of multiple
factors that determine the concentrations and/or activities of the
proteases that degrade oxidatively damaged protein, on the other. As
illustrated in Fig. 4, many different
physiological and environmental processes lead to the formation of ROS.
Collectively, these processes can promote the generation of a battery
of ROS, including a number of free radicals (·OH, O2,
R·, ROO·, RO·, NO·, RS·,
ROS·, RSOO·, and RSSR
), various non-radical
oxygen derivatives (H2O2, ROOH, 1O2, O3, HOCl, ONOO
,
O=NOCO2
,
O2NOCO2
,
N2O2, NO2+, and
highly reactive lipid- or carbohydrate-derived carbonyl compounds,
viz. 4-hydroxy-2-nonenal, malondialdehyde ketoamines, ketoaldehydes, and deoxyosones. Any one of these ROS is capable of
promoting the modification of proteins. However, as shown in Fig. 4,
their abilities to do so are dependent upon the concentrations of a
myriad of enzymic and non-enzymic factors (antioxidants) that can
either inhibit the formation of ROS or facilitate their conversion to
inactive derivatives. For example, the O
2 formed by several
pro-oxidant systems shown in Fig. 4 is readily converted to
H2O2 by the action of superoxide dismutase.
This H2O2 together with
H2O2 produced by various oxidases and
metal-catalyzed oxidation systems is readily degraded by catalase,
glutathione peroxidase, thiol-specific antioxidant enzymes, and other
peroxidases. However, if in the course of metabolism the concentrations
of these antioxidant activities become insufficient to decompose all of
the H2O2 formed, the
H2O2 may undergo metal ion-catalyzed cleavage
by the Fenton reaction to generate the even more toxic ·OH. This
reaction is dependent upon the availability of iron and copper, which
is determined by the concentrations of metal-binding proteins
(ferritin, transferrin, lactoferrin, and ceruloplasmin), and of
multiple factors (iron-responsive elements, etc.) that control the
intracellular concentrations of these proteins, as well as factors that
influence the binding and/or the release of metal ions from these
binding proteins. The level of ROS is also a function of the
concentrations of vitamins (A, C, and E) and of metabolites (uric acid,
bilirubin, etc.) that are capable of either scavenging free radicals
directly or of facilitating the regeneration of metabolites that do so.
Finally, metal ion chelators can either suppress or enhance the rates
of ROS generation by forming complexes with iron or copper that inhibit
their ability to catalyze ROS formation or alter their redox potentials
and therefore their ability to undergo cyclic interconversion between oxidized and reduced states. Furthermore, other divalent cations (Mg2+, Mn2+, and Zn2+) may compete with Fe(II)
or Cu(I) for binding to metal binding sites on proteins and thereby
prevent site-specific generation of ·OH, which is likely the
most important mechanism of protein damage (42). In addition, Mn(II) is
able to inhibit the reduction of Fe(III) to Fe(II) (43) and thus
prevent its ability to promote formation of ·OH by the Fenton
reaction as well as the generation of other forms of ROS as illustrated
in Fig. 1.
As noted above, the accumulation of oxidized protein reflects not only the rate of protein oxidation but also the rate of oxidized protein degradation, which (as shown in Fig. 4) is also dependent upon many variables, including the concentrations of proteases that preferentially degrade oxidized proteins and numerous factors (metal ions, inhibitors, activators, and regulatory proteins) that affect their proteolytic activities. For example, oxidized forms of some proteins (e.g. cross-linked proteins (44-46)) and proteins modified by glycation (47) or lipid peroxidation products (44) are not only resistant to proteolysis but, in fact, can inhibit the ability of proteases to degrade the oxidized forms of other proteins (44, 48).
The foregoing discussion and some of the points illustrated in Fig. 4
are by no means comprehensive. They are intended to call attention to
the extraordinary complexity of ROS biochemistry. Numerous other
factors not discussed are certainly important in determining the
steady-state level of oxidative damage under varying physiological and
environmental conditions. It is our belief that during aging there is a
progressive accumulation of errors at the level of DNA that affect any
one or more of the factors that govern the dynamics of protein
oxidation and oxidized protein degradation. This leads to a shift in
the balance between these processes in favor of oxidized protein
accumulation and attendant loss of biological function. Two
observations are consistent with this hypothesis. (a) The
level of oxidized protein in cultured fibroblasts is a function of the
age of the fibroblast donor and is independent of the cell passage
number. (b) Chronic injection of the free radical scavenger
tert-butyl--phenylnitrone leads to a reversal of some
age-related changes in the gerbil brain, but when the
tert-butyl-
-phenylnitrone treatment is discontinued the
age-related changes reappear (36). Both observations indicate that the
level of oxidative damage is determined by the genetic make-up of the
cell, which changes with age. According to this proposition, the aged
phenotype could be expressed (a) by a single point mutation
that could impair a biological activity that occupies a central role in
biological functions, such an alteration of helicase as occurs in
Werner's syndrome (49), or (b) by the accumulation over
time of numerous errors leading to deficiencies in the synthesis and/or
activities of a multiplicity of the factors that govern the balance
between protein oxidation and degradation. From this perspective, aging
could be looked upon as a degenerative process (disease?) that
might include aberrations that contribute to the development of other
pathologies, such as Alzheimer's disease, amyotrophic lateral
sclerosis, diabetes, etc., in which the accumulation of oxidatively
modified protein has been demonstrated. Perhaps in these diseases one
or more of the specific processes summarized in Fig. 4 are exaggerated,
leading to unique manifestations that are characteristic of the
disorder.