Sci. Aging Knowl. Environ., 17 March 2004
Vol. 2004, Issue 11, p. pe12
[DOI: 10.1126/sageke.2004.11.pe12]


Exploiting Proteomics in the Discovery of Drugs That Target Mitochondrial Oxidative Damage

Bradford W. Gibson

The author is at the Buck Institute for Age Research, Novato, CA 94945, USA and in the Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA. E-mail: bgibson{at}

Key Words: mass spectrometry • neurodegenerative disease • mitochondrial complex I • oxidative stress • posttranslational modifications


By the measure of popular culture, one might conclude that the use of antioxidants to treat aging and age-related diseases was settled science. One has only to turn on the television, surf the Web, or flip through the pages of most magazines to encounter any number of advertisements with claims that antioxidants will slow or even reverse aging. Not since Linus Pauling first touted the virtues of large doses of vitamin C as a potent antioxidant to treat cancer (1) and increase longevity (2) have we witnessed such an explosion in the interest of antioxidants as drugs and dietary supplements. By now, most of us are aware that foods ranging from blueberries to red wine to green tea contain potentially important compounds that can scavenge free radicals or other reactive oxygen species (ROS). The active components in these foods include polyphenolics, flavanoids, and carotenoids, to name only a few. What's missing of course, at least to scientists, is a significant body of rigorous clinical studies to support the claims that antioxidants are protective against age-related diseases or can increase life span to any significant extent. A recent report from Johns Hopkins University showing a reduced risk of Alzheimer's disease in users of the antioxidant vitamins C and E, but only when given together (3), provides some important movement toward this goal, but also suggests that the mechanisms that underlie the therapeutic effects of these and other antioxidants are likely to be more complex than originally thought.

As a chemist who has taught drug courses to pharmacy students for almost 20 years and has recently joined a new institute whose scientific mission is to understand the molecular mechanisms of aging and age-related diseases, it is interesting to consider what new developments might propel the science of antioxidant drug therapy to a more serious level of inquiry. In particular, the rapid advance of proteomic methodologies and their application to large-scale studies of protein-protein interactions and protein expression profiles (4, 5) suggest that these methods are well suited to provide the molecular details needed to truly understand oxidative injury. Global proteomic analysis, for example, could be used to better assess the efficacy of antioxidant drug therapies by cataloging changes in protein expression or identifying proteins that undergo structural modifications, especially ones that are caused by oxidative damage.

Oxidative Damage to Proteins

A number of macromolecules, including proteins, DNA, and lipids, are modified under conditions of oxidative stress (Fig. 1). In proteins, these modifications may be part of complex signaling pathways that sense changes in the redox environment as a component of their regulatory control, as in S-nitrosylation (6) or the oxidation of cysteine to sulfenic acid (7, 8). The majority of these oxidative modifications, however, are not regulatory per se but are rather undesired products of reactions involving ROS, which are thought to contribute to aging and various neurodegenerative diseases (9-12). A shift to oxidative conditions and the modifications that result can seriously compromise a protein's function or structure, leading to increased hydrophobicity and protein turnover, cross-linking, or aggregation (13, 14). The ROS thought to mediate (or serve as precursors to) these latter types of protein modifications include hydrogen peroxide (HOOH), superoxide anion (O2·-), or hydroxyl radicals (HO·), although other species, such as the endogenous aldehydes 4-hydroxynonenal and malondialdehyde (15, 16), or reactive nitrogen species such as peroxynitrite (_ONO2) (17) are also likely to be key players.

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Fig. 1. Oxidative stress can increase through the action of endogenous as well as exogenous factors, giving rise to a number of ROS or their precursors (NO, for example). Although protective mechanisms exist to regulate these species, damage can occur, leading to both reversible and irreversible oxidative damage to proteins, DNA, and lipids. Under conditions of elevated oxidative stress, perhaps as cells age or in various disease states, these oxidative lesions can accumulate and have drastic consequences for cellular function, leading ultimately to senescence, transformation, or cell death. SOD, superoxide dismutase; CoQ10, coenzyme Q10; HSPs, heat shock proteins; NOS, nitric oxide synthase; 2dG, 2-deoxyglucose; BSO, butamine sulfoximine; perox., peroxidase.

The amino acids that are typically involved in oxidative modification are surprisingly broad in their functionalities (Fig. 2) (18). Simple deamidation reactions can occur to asparagine and glutamine by nonenzymatic hydrolysis to the corresponding acids or related compounds (19). Nucleophilic groups such as the {epsilon}-amino group of lysine, the sulfhydryl of cysteine, or the imidazole nitrogen of histidine can react with various aldehydes to form stable Michael addition-type products (15, 16). Alternatively, direct attack by hydroxyl radicals at lysine, serine, proline, and arginine side chains has been shown to give rise to a series of semialdehydes and ketones, including {alpha}-aminoadipic semialdehyde, glutamic semialdehyde, and 2-pyrrolidone (20). Aromatic amino acids, tyrosine in particular, are prone to aromatic nitration reactions and can form highly stable nitroaromatic end products such as 3-nitrotyrosine (17). The indole ring of tryptophan can add one or two oxygens to yield the open-ringed N-formylkynurenine product. Methionine is readily oxidized to the sulfoxide, but this can be reversed in vivo through the action of endogenous methionine reductases (21). Cysteine residues are perhaps the most sensitive to oxidation, yielding glutathione adducts (_SSG), disulfide bonds (_S-S-), nitrosylated thiols (_S-NO), or the oxygenated acids sulfenic (_SOH), sulfinic (_SO2H), and sulfonic (_SO3H) acids. Some of these modifications are reversible in vivo, but many are not. Although evidence is mounting that these protein modifications are damaging and possibly cumulative in some cases, there is surprisingly little data (albeit with a few exceptions) on specific proteins that undergo oxidation as well as the precise sites within these proteins that are modified. Without the molecular details of how these oxidative modifications occur throughout all proteins within individual tissues or cells or their subcellular compartments (the "proteome"), it is difficult to draw many conclusions about their overall functional importance.

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Fig. 2. Some amino acid side-chain products of oxidative damage. (A) Addition of oxygen to methionine to form the sulfoxide [Met(O)]; (B) glutathione adduction to cysteine (Cys-SG); (C) aromatic nitration of tyrosine to form 3-nitrotyrosine (3-NTyr); (D) two-oxygen addition to tryptophan to yield N-formylkynurenine [Trp(O2)]; (E) oxygen addition to lysine followed by loss of ammonia to yield {alpha}-aminoadipic semialdehyde (AASA); (F) Michael addition of 4-hydroxynonenal to cysteine (Cys-HNE).

The Mitochondrion as a Target for Antioxidant Therapy

It has been suggested that mitochondria are a desirable pharmacological target (22), and drugs that modulate mitochondrial function include those that target oxidative stress (23). One reason why this may be true is the crucial role mitochondria play in energy metabolism and cell death signaling pathways, both of which have links to cancer, neurodegenerative disease, diabetes, and aging (see "The Two Faces of Oxygen" and the Nicholls Perspective) (24). These organelles are also the source of superoxide radical anions, which arise from oxidative phosphorylation and can in turn lead to the production of ROS. Although the cell has evolved mechanisms to deal with the harmful effects of superoxide, including the enzymes superoxide dismutase, catalase, and glutathione peroxidase, these safeguards do not completely prevent the formation of ROS, particularly under conditions of elevated or prolonged stress. Given their close proximity to these ROS, mitochondrial proteins would be expected to be among the most likely targets of oxidative damage, especially for highly reactive species whose reactions are diffusion-controlled.

Current drug strategies that selectively target mitochondria take advantage of the electrochemical gradient from the outer plasma membrane ({Delta}{Psi}p = 30 to 60 mV) to the inner mitochondrial matrix ({Delta}{Psi}m = 150 to 180 mV). This gradient provides a large driving force for the selective targeting and concentration of large lipophilic cations to the mitochondria (22, 23, 25). Two drugs of this type, MitoQ and MitoVitE, have been proposed by Murphy and colleagues to treat Friedreich's ataxia (26). Both compounds are based on triphenylphosphonium modifications to coenzyme Q and vitamin E, respectively (Fig. 3). In these studies, MitoVitE was found to be 350 times more potent than Trolox, a water-soluble version of vitamin E, and MitoQ was 100 times more potent than idebenone, a coenzyme Q derivative. In a separate study, Melov and Lithgow (27) fed superoxide dismutase-catalase mimetics to Caenorhabditis elegans, which then showed a significant increase in life span as compared to wild-type organisms. They proposed that these mimetics increased life span by augmenting natural antioxidant defenses, supporting the notion that oxidative stress is an important determinant of the aging rate. However, in another study, worms treated with the Euk-8 compound showed a reduction in mean life span. These opposing observations may have resulted from the different methods used to administer the drug (daily versus every-other-day treatment) and/or the different compound sources.

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Fig. 3. Various compounds that target oxidative stress in mitochondria. Coenzyme Q and vitamin E are natural compounds, both of which are used as dietary supplements. The analogs of these two compounds, MitoQ and MitoVitE (26), have been redesigned to improve uptake and increase target selectivity to mitochondria. The company Eukarion has developed mimetics of superoxide dismutase and catalase (Euk-8, Euk-134, and Euk-189) (70), several of which were shown to dramatically increase the life span of wild-type C. elegans (27) (and see "Wrinkle Treatment for Worms"), as well as being effective in a mammalian model of mitochondrial oxidative stress (see "Drugs Protect Mice From Pernicious Forms of Oxygen") (30). Flupirtine is a nonopioid analgesic drug with mitochondrial antioxidant activity.

An analog of the superoxide dismutase-catalase mimetics was also shown to be effective in a direct test of mammalian mitochondrial oxidative stress in an animal that lacks mitochondrial SOD, the sod2 knockout mouse (28, 29) (see "Drugs Protect Mice From Pernicious Forms of Oxygen"). Beneficial effects were observed in the brain and heart and in a number of other tissues (30, 31). And in a final example, flupirtine, a nonopioid analgesic that also possesses potent antioxidant activities, was recently found to preferentially localize to the mitochondria, where it can scavenge free radicals (32). Like other drugs that target the mitochondria, the selective action of flupirtine is presumably a result of its cationic and lipophilic properties. Curiously, flupirtine has been shown to possess activities that inhibit ischemic injury (33) and apoptosis, and in a more controversial role, may be protective against Alzheimer's and prion diseases (34).

The Current Status of the Human Mitochondrial Proteome

Over the past couple of decades, considerable success has been made in identifying individual proteins that are localized to the mitochondria, in particular the 100 or so subunits that constitute the five complexes of the electron transport chain (ETC). The majority of these data came originally from mitochondria isolated from bovine heart tissue, as best exemplified by work from Walker's group at the Medical Research Council in Cambridge. Decades worth of research by this group led to the identification of 45 subunits in bovine complex I (35) and to a crystal structure of the F1-adenosine triphosphatase (ATPase) (complex V), which consists of eight subunits ({alpha}3{beta}2{gamma}{delta}{epsilon}) that are bound to the ATPase inhibitory protein IF1 in the presence of ATP (36).

More recently, using modern mass spectrometry (MS)-based proteomic strategies, several groups have begun to tackle the larger job of determining the composition of entire mitochondrial proteomes from a number of important model systems, including yeast (37), mouse (38), and rat (38-40). MS was a critical component in all these analyses, because it allowed these researchers to identify hundreds of unique proteins from mass spectral data on small peptides derived from these proteins. The peptides can be acquired in a high-throughput manner after proteolytic digestion. In probably the most ambitious study to date, Mootha and colleagues (38) identified 591 mitochondrial proteins from mouse brain, heart, kidney, and liver, 163 of which had not been previously associated with the mitochondria. Although studies of this type are no doubt critical to understanding the nature of this organelle and its contribution to disease, they do not diminish the need to identify mitochondrial proteins from human sources, because these will be the ones that are eventually targeted in any future drug discovery initiative.

In one of the first MS studies targeting the human mitochondrial proteome, proteins isolated from human placental mitochondria were first analyzed by two-dimensional gel electrophoresis (2DE), which separates proteins on the basis of charge (isoelectric focusing) and size [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)]. After straining, individual proteins (that is, spots) were then cut out from the 2DE gel and analyzed by matrix-assisted laser desorption ionization (MALDI) MS after tryptic digestion (41). Although only 46 proteins were identified in this initial effort, this total increased incrementally over the past few years when multiple, more narrow-range isoelectric focusing gradients were used (42). In a separate study, we prepared highly purified mitochondria from a human SH-SY5Y neuroblastoma cell line by metrizamide gradient centrifugation and identified 61 of the 84 most abundant protein spots after 2D gel separation (43). One advantage of this study over the placental initiative was the absence of any significant protein contamination from other cellular compartments or organelles. However, like the placental study, the neuroblastoma cell line study was rather limited in protein coverage and identified only 10 to 20% of the 1500 proteins estimated to be contained in this organelle (44). In particular, small, basic, and/or very hydrophobic proteins were significantly underrepresented, which are classes of proteins known to be present in relatively high abundance in the mitochondria.

In an effort to overcome the limitations of 2D gel separation and to substantially increase the overall proteome coverage, we obtained mitochondria from human heart tissue and shifted our analytical protein separation scheme away from the 2D gel protocols to a combined sucrose gradient fractionation and 1D SDS-PAGE separation scheme (45). This change of strategy was fruitful, and, to date, 684 unique proteins have been identified from the combined peptide data obtained from over 100,000 mass spectra generated by MALDI-MS and high-performance liquid chromatography (HPLC) MS/MS analyses (46-48) (Fig. 4). These data are now part of MitoProteome, a publicly accessible database for the human heart mitochondrial proteome (49). This database joins MITOP, an earlier attempt at identifying human mitochondrial genes and proteins that contains over 340 unique entries (50).

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Fig. 4. Classification of proteins identified in human heart mitochondria using high-sucrose-gradient fractionation, 1D gel separation, and HPLC MS/MS analysis (47). This classification includes 498 of 615 of the total proteins identified by MS, leaving 19% without identifiable or putative functions. An update of the proteome includes 253 proteins identified using multidimensional chromatography protein identification technology (also referred to as MudPIT) (71), bringing the new total to 684 unique proteins (48). TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation.

Despite the progress made in these combined efforts, the human mitochondrial databases have not yet been exploited to any great degree in identifying or targeting new candidates for drug development. Nor have these proteomes been used to design mitochondrial protein biomarkers that might be useful in drug screening or clinical diagnosis. Given the broad functional classes that have been identified so far (for example, proteases, signaling proteins, and transport proteins), it seems to be only a matter of time before the mitochondrial proteome is exploited in drug development, especially when the growing numbers (now over 100) of still unclassified proteins are ultimately assigned functions (Fig. 4).

Proteomic Approaches for Assessing the Extent of Oxidative Damage and Identifying Therapeutic Targets

Antibodies directed at specific functional groups have been the primary means to assess the presence and extent of protein oxidative damage and/or modifications. For example, both mono- and polyclonal antibodies are commercially available for the detection of the following modifications in proteins: 3-nitrotyrosine, carbonyl and aldehyde adducts, and S-nitrosylation. In some cases, antibodies have been made against sequence-specific modifications, such as those directed at nitration of a select group of tyrosine residues in {alpha}-synuclein, a protein that can carry certain mutations associated with Parkinson's disease (51). Coupling an immunological assay with 1D and/or 2D gel separation followed by MS identification has been used in recent years to provide information related to the extent of oxidative damage in proteins, although these methods are time-consuming and have rarely led to the identification of the precise amino acid sites at which the oxidative lesions occur.

At the Buck Institute, several proteomic projects are in progress that are examining proteins involved in Parkinson's and Alzheimer's diseases and several animal models of aging. One common feature of these investigations is to identify proteins that have undergone oxidative modification, as well as the molecular and site-specific details of these oxidative events. For example, in a recent paper by Andersen and colleagues (52), the activity of mitochondrial complex 1 was seen to specifically decrease in a rodent PC12 dopaminergic cell line when the total pools of the antioxidant glutathione were lowered (see Andersen Review). In this model of Parkinson's disease, glutathione concentrations were reduced in an inducible manner by the addition of doxycycline, which controlled the expression of antisense messages that blocked the synthesis of {gamma}-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis. The loss of activity of complex I was recoverable by treatment with the antioxidant dithiothreitol, suggesting that one or more key cysteine residues in the complex I proteins were oxidized (and thus inactivated) in the presence of low glutathione concentrations. The recent availability of a monoclonal antibody that allows rapid one-step purification of this complex (53) has greatly aided our continuing efforts to obtain sufficiently purified material with minimal workup to identify the protein subunit(s) and modification sites involved. Nonetheless, the challenge is considerable: 35 of the known 45 subunits in complex I contain at least one cysteine, with the 75-kD subunit (Ndufs1) alone containing 18 such residues.

As part of our effort toward understanding the molecular basis of the selective oxidation of complex I, we recently proposed a method that uses common alkylating reagents to determine the redox state of individual thiols in proteins (54) (Fig. 5). The essential idea is to label the thiols that are readily accessible with alkylating reagents containing several stable isotopes (for example, N-ethylmaleimide or iodoacetic acid), followed by denaturation, reduction, and complete labeling of the remaining thiols with the same reagent in its normal isotopic form. After proteolysis of the fully alkylated protein(s), peptides containing cysteines will appear as doublets in the mass spectra, separated in mass by the number of C13 atoms in the alkyating reagents, if not fully alkylated in the first step (that is, before denaturation and reduction.) Therefore, the ratio of the two isotopic forms can yield information about the redox state of the individual cysteines in the target protein. This procedure, which can be referred to as "differential alkylation," is analogous to isotope-coded affinity tag (ICAT) methods (55), which differentially label cysteines in two separate protein pools that are undergoing direct comparison for the determination of their relative expression levels. Indeed, another group has recently published a method similar to our own protocol, using an acid-cleavable ICAT reagent that has nine C13 atoms in the linker region and a biotin group for affinity purification (56). In addition, Murphy's group has described reagents to be used for a purpose similar to that of the ICAT reagent (57), but Murphy's agents contain a triphenylphosphonium tag, which localizes to the mitochondria and therefore allows one to target thiol groups in mitochondrial proteins even when the cell or the organelle is intact and functional. An intriguing report from this group (58) showed that ROS production by mitochondrial complex I increased when the glutathione pools were shifted in favor of oxidized glutathione (GSSG), resulting in specific glutathiolation of the 51- and 75-kD subunits of complex I.

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Fig. 5. A proposed differential alkylation method for assessing the redox status or oxidative damage of specific thiol groups in proteins. (Top) The sulfhydryl groups of cysteine residues can undergo a series of oxidative modifications, including glutathiolation, intramolecular disulfide formation, nitrosylation, and oxidation to sulfonic acid, among others. (Bottom) As we originally proposed (54), proteins containing oxidized cysteines can be first labeled by S-alkylation, followed by reduction and denaturation of the protein(s), and final labeling of the remaining, now reduced, cysteines, with a stable isotope version of the same alkylating reagent. In our case, both N-ethylmaleimide and iodoacetic acid were used, both containing two C13 atoms. Alternatively, one can use an acid-cleavable ICAT reagent containing nine C13 atoms and a biotin group that allows for subsequent enrichment, as proposed by Sethuraman et al. (56).

Finally, Melov and colleagues at the Buck Institute are in the process of examining proteomic targets of mitochondrial oxidative stress in their mitochondrial Sod2 null mice model. This group has recently published a study in which they examined the activities of the ETC complexes and the tricarboxylic acid cycle enzymes {alpha}-ketoglutarate dehydrogenase and citrate synthase in the Sod2 null mice (31). One particularly intriguing observation was the differential sensitivities of these mitochondrial proteins to oxidative stress and the fact that antioxidant treatment could rescue the neuronal cell death phenotype.

In addition to determining thiol redox states, proteomic-wide mass spectrometric approaches have been applied to identify mitochondrial proteins containing 3-nitrotyrosine and oxidized tryptophan modifications. Several years ago, my colleagues and I and another group separately described a MALDI-MS approach that relied on identifying peptides that contain 3-nitrotyrosine by the presence of a unique molecular ion signature arising from a photodecomposition reaction involving the loss of one and two oxygen atoms (59, 60). Using this approach, we are currently examining a series of proteins prepared by immunoprecipitation of individual ETC complexes that show positive staining with antibodies to 3-nitrotyrosine (61). In an alternative approach, high-throughput electrospray ionization HPLC-MS/MS analysis was combined with immunological staining to identify nitrated tyrosine modifications among mitochondria proteins separated by sucrose gradient fractionation (62). This method revealed that complex I subunits, particularly Ndufb4 and Ndufa6, contain the highest degree of nitrated products in the mitochondrial proteome. And in cases where selected molecular ions for individual peptides were subjected to gas phase fragmentation and their products analyzed in a second mass spectrometer (MS/MS), specific tyrosines in several subunits of complex I were identified as the nitrated sites. In a second mass-spectrometric study by this same group (63), tryptophan oxidation was also investigated among mitochondrial proteins through the identification of the two-oxygenated product N-formylkynurenine. Subunits from complexes I and V and other proteins involved in redox metabolism were found to be especially sensitive to oxidation in mitochondria obtained from pooled heart donors.

The possibility of designing new chemical strategies or multiplexing MS/MS experiments against a wide range of modifications would be particularly attractive, because the new methods would be expected to both conserve the sample and provide a more comprehensive look at protein oxidative damage. Toward this goal, Ahmed et al. (64) used MS-based multiple reaction monitoring to examine 16 biomarkers, including those for nitrosylation, glycation, and other oxidation products. Although there are well-established methods for determining the extent of carbonyl modifications in proteins (65), newer methods have been proposed that are suitable for large protein mixtures, such as that described by Soreghan and colleagues. This new method allowed the high-throughput analysis of carbonylated proteins from mouse brain tissue using a hydrazide biotin-streptavidin enrichment strategy, followed by HPLC-MS/MS (66). In this latter study, it is interesting to note that a number of mitochondrial proteins were among those identified as being carbonylated, including three that are involved in ATP production (ATPase, isocitrate dehydrogenase, and succinyl-CoA ligase).

What Can We Expect from Proteomics in the Near Future?

Proteomics seems poised to tackle some of the more difficult challenges inherent in analyzing global protein modifications (67). New approaches have been advanced in the past year or two that target, for example, proteome-wide changes in phosphorylation (68) and ubiquitination (69). The challenge remains, however, to develop more appropriate proteome-wide strategies that target oxidative modifications, especially enrichment techniques capable of handling the diverse set of products that encompass protein oxidation reactions. MS, perhaps coupled to immunochemical approaches, could be better optimized to perform this function. As an alternative, the development of selective chemical tools in conjunction with high-throughput MS may represent a fruitful approach. In any case, obtaining a proteomic analysis of oxidative stress and having the means to evaluate the effects of antioxidants on preventing or modulating oxidative modifications will hopefully lead to a better understanding of the molecular basis of oxidative damage and assessment of antioxidant therapy.

March 17, 2004
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Citation: B. W. Gibson, Exploiting Proteomics in the Discovery of Drugs That Target Mitochondrial Oxidative Damage. Sci. Aging Knowl. Environ. 2004 (11), pe12 (2004).

Science of Aging Knowledge Environment. ISSN 1539-6150