96 L.V. Thompson 6.4 ROS Attack: Proteins Almost all amino acid residues in a protein can be oxidized by ROS. Oxidative products of amino acid residues include the formation of disulfide bonds at cysteine residues, carbonyl derivatives, and many others oxidized residues, such as methionine sulfoxide. These oxidative modifications lead to functional changes in various types of proteins, which have substantial physiological impact. For instance, oxidative damage to enzymes causes a modification of their activity, while oxidant-derived injury to structural proteins and chaperones produce protein aggregation. Specifically, the accumulation of damaged proteins is dependent upon the balance between many different processes including: (1) the rate of ROS synthesis by any one of the numerous mechanisms; (2) the ability of various antioxidants to scavenge ROS; (3) the ability to repair nucleic acid damage leading to generation of altered proteins that are highly sensitive to oxidation; (4) the concentrations of proteases that degrade oxidized forms of proteins); (5) the generation of cross-linked proteins that inhibit the proteolytic degradation of oxidized proteins; (6) and the ability to repair oxidation of sulfur-containing amino acid residues of proteins. 6.5 4-Hydroxy-2-nonenal (HNE) Mechanisms of damage and/or cell signaling can be direct, for example through the effects of superoxide, or can be introduced into proteins by reaction with aldehydes formed during lipid peroxidation (e.g. 4-hydroxy-2-nonenal or malondialdehyde Fig. 12 Oxidative Stress. ‘Oxidative stress’ is a disturbance in the prooxidant–antioxidant balance in favor of prooxidant, leading to oxidative damage. Increased levels of reactive oxygen species can directly or indirectly damage macromolecules such as phospholipids, nucleic acids, and proteins. In the skeletal muscle sarcomere, increased damage to actin and myosin has potential to interrupt actomyosin interaction resulting in skeletal muscle contractility deterioration 97Age-Related Decline in Actomyosin Structure and Function that react with the є-amino group). 4-hydroxy-2-nonenal, HNE, is a reactive aldehyde that originates from the peroxidation of membranes and forms a mixture of adduct types on the side-chains of cysteine, lysine, and histidine through a Michael-type nucleophilic addition. The HNE adducts may inhibit protein function. For example, the adenine nucleotide transporter is particularly susceptible, as is the matrix enzyme, aconitase (Yan and Sohal 1998). In both, the degree of damage (measured as protein carbonyls) is correlated with the loss of protein function (Yan and Sohal 1998). 6.6 3-Nitrotyrosine (3-NT) 3-nitrotyrosine (3-NT) has been identified as a stable marker of protein oxidative damage. This post-translational chemical modification can alter protein function and is associated with acute and chronic disease states. 3-nitrotyrosine, 3-NT, is formed when tyrosine is nitrated by peroxynitrite, a highly reactive molecule gener- ated by the reaction of nitric oxide with superoxide. During muscle contraction the individual fibers are exposed to periodic fluxes of nitric oxide and superoxide lead- ing to favorable conditions for the formation of peroxynitrite. Tyrosine nitration has been shown to inhibit protein function by altering a protein’s conformation, impos- ing steric restrictions to the catalytic site, and preventing tyrosine phosphorylation (Cassina et al. 2000). Taken together, the functional significance of tyrosine nitra- tion depends on two factors (1) the site of modification and (2) the extent of the protein population containing functionally significant modifications. 6.7 Oxidative Stress and Muscle Dysfunction Skeletal muscle is vulnerable to oxidative stress for several reasons. First, skeletal muscle proteins are exposed to stress during contraction because there is rapid and coordinated changes in energy supply and oxygen flux. Subsequently, there is an increase in electron flux and leakage from the mitochondrial electron transport chain. Second, the high concentration of myoglobin within skeletal muscle also plays a role because the heme-containing protein is known to confer greater sensitivity to free radical-induced damage to surrounding macromolecules by converting hydrogen peroxide to other more highly reactive oxygen species (Ostdal et al. 1997). Skeletal muscle fiber-type differences in susceptibility to oxidative stress may be mechanistically related to the aging phenotype discussed earlier in the chapter (both fiber types show susceptibility to age-related dysfunction, but the time course of change is fiber type-dependent). There are fundamental metabolic differences between slow-twitch aerobic fibers and fast-twitch glycolytic fibers. In particular, 98 L.V. Thompson the major energy pathway utilized in type I fibers occurs through oxidative metabolism, whereas the glycolytic pathway is the primary means for generating energy in type II fibers. Thus, type I fibers likely produce greater ROS via mito- chondrial oxidative phosphorylation compared with type II fibers. To counter the effects of ROS, type I fibers have higher antioxidant capacities that prevent or attenuate oxidative damage (Ji et al. 1998; Ji 2001, 2002; Reid and Durham 2002). Although type II fibers may generate lower levels of ROS during metabolism than do type I fibers, type II fibers may be more susceptible to oxidative stress because their antioxidant defenses are less robust. 7 Age-Related Post-translational Modifications of Proteins 7.1 Aging With aging, under basal skeletal muscle conditions oxidant production is increased and the redox state shifts to a more oxidative environment (Ji et al. 1998; Ji 2001). While some antioxidants are increased in aging skeletal muscle, the extent of increase is muscle-specific and not global to all enzymes. Thus, the burden of defending against the increased load of free radicals may be greater than the com- pensatory change in antioxidants. If the antioxidant system is inadequate and key skeletal muscle proteins are modified, the proteasome must remove damaged pro- teins (one of the major degradation pathways for damaged proteins in skeletal muscle). Yet, the proteasome function in muscle declines with aging (Husom et al. 2004, 2005). Thus, the fundamental changes in cell redox status and the ability to remove free radical damaged proteins likely contribute to the age-related changes in muscle contractility discussed above. 7.2 Myosin and Actin – Key Contractile Proteins and Post-translational Modifications As discussed earlier, in aged muscle, there is a reduction in the fraction of myosin heads in the strong-binding structural state, such that there are fewer myosin-actin interactions capable of generating force (Fig. 8). In addition, a significant age- related inhibition of myosin ATPase, critical for generating force, is reported from investigations of isolated proteins (myosin and actin) (Fig. 9). Thus, mechanisms that decrease or interrupt the interaction of myosin and actin are likely to explain the age-related reduction in force-generating capacity. One mechanism that may play a role in the age-related decline in contractility (e.g., interrupt the interaction of myosin and actin) is an accumulation of damage from post-translational chemical modifications (e.g., oxidative damage) to myofibrillar 99Age-Related Decline in Actomyosin Structure and Function proteins (Fig. 12) because in vitro studies demonstrate that peroxynitrite impairs both energetics and contractility of permeabilized muscle fibers (Callahan et al. 2001). Age-related oxidative damage of myosin and actin are probably accumu- lating in muscle as a consequence of decreased muscle protein turnover (Balagopal et al. 1997; Ferrington et al. 2005; Husom et al. 2005). To date, studies of in vivo oxidative modifications of myosin and actin focus on selective markers of oxidative damage such as nitration, formation of HNE adducts, oxidation of cysteines and glycation. In a systematic study of in vivo oxidative modifications of myosin and actin and aging, both nitration and the formation of HNE adducts were evaluated (Thompson et al. 2006). The levels of these two markers of oxidative stress, 3-nitrotyrosine and HNE-adducts, on myosin and actin did not increase with age. This finding suggests that accumulation of oxidative damage to these two key myofibrillar proteins does not occur with age. In contrast to the similar amounts of 3-nitrotyrosine and HNE-adducts on myosin and actin with age, the results of several other investigations suggest that actin and myosin have protein-specific differences in susceptibility to oxidation (Kaldor and Min 1975; Prochniewicz et al. 2005; Srivastava and Kanungo 1982). Studies on purified proteins show an age-related decrease in cysteine content in myosin, but cysteine content of actin is unaffected by age (Prochniewicz et al. 2005). The implication of this finding for muscle contractility depends on the still unknown localization of oxidized sites. Oxidation of one or two reactive myosin cysteines (Cys 707 and Cys 696) could result in significant deterioration of muscle contractility, but myosin contains about 40 cysteines, and the functional role of the majority is not known (Bobkov et al. 1997; Crowder and Cooke 1984). 7.3 SERCA and Post Translational Modifications The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a membrane protein responsible for the active transport of calcium from the cytosol into the sarcoplas- mic reticulum lumen, thus removing Ca 2+ from the vicinity of the contractile pro- teins and causing muscle relaxation. Therefore, changes in the Ca-ATPase function have a direct impact on muscle performance. Ca-ATPase function decreases in an age-dependent manner. The SERCA protein is probably the most extensively investigated muscle pro- tein, from a biochemical perspective with aging. These investigations focus on what sites are vulnerable to oxidative stress, and how the modification or damage alters protein function with increasing age. Normal aging of skeletal muscle is associated with increased nitration; in particular, specific nitration of the SERCA2a isoform in slow-twitch muscle (Viner et al. 1999). Tyrosine nitration increases by at least threefold in skeletal muscle during normal aging, and cor- relates with a 40% loss in Ca 2+ -ATPase activity during normal aging. Mass spec- trometry analysis reveals an age-dependent accumulation of 3-NT at positions 100 L.V. Thompson 294 and 295 of the SERCA2 protein, suggesting that these tyrosines play a critical role in muscle function. In vitro studies also demonstrate that SERCA2a is inherently sensitive to tyrosine nitration with concomitant functional deficits. Because the physiological role of the Ca-ATPase is to mediate muscle relaxation, the consequence of nitration-induced inhibition of SERCA2a most likely explains the slower contraction and relaxation times observed in skeletal muscle with normal aging. Aging also leads to a partial loss of SERCA1 isoform activity, and a molecular rationale for this phenomenon may be the age-dependent oxidation of specific cysteine residues (Viner 1999a, b). Mapping of the specific cysteine residues reveals nine cysteine residues targeted by age-dependent oxidation in vivo, and six cysteine residues partially lost upon oxidant treatment in vitro. Interestingly, the residues affected in vivo do not completely match those targeted in vitro, suggest- ing that modification of some residues do not contribute significantly to the loss of SERCA function with age. Taken together, these studies provide some insights about the molecular mechanisms responsible for age-related alterations in calcium regulation in skeletal muscle. 7.4 Aging Skeletal Muscle Phenotype – Nitration and Skeletal Muscle Proteins One goal of global proteomic experiments in the field of aging is the identification and functional characterization of post-translationally modified proteins in vivo, and to determine whether such modifications are mechanistically related to specific aging phenotypes. The recent development of high resolution separation techniques and mass spectrometry (MS) instrumentation permits the identification of function- ally important post-translational protein modifications occurring during aging. In order to evaluate the role of oxidative stress and the skeletal muscle aging phenotype, comparison of damaged skeletal muscle proteins in two muscles, the soleus and semimembranosus, each composed of different skeletal muscle fiber types (Fugere et al. 2006). Specifically, the soleus muscle is composed of >90% type I fibers, whereas the semimembranosus is composed of >90% type IIB fibers. In these series of experiments, it was hypothesized that with aging the semimem- branosus (type II) muscle would accumulate a greater amount of protein tyrosine nitration compared to proteins in the soleus (type I) muscle (Fugere et al. 2006). Previous in vitro studies show impairment in both energetic and contractility when permeabilized skeletal muscle fibers were exposed to peroxynitrite (Callahan et al. 2001). Moreover, the extent of functional decline is consistent with age-induced changes in single fiber contractile properties, suggesting that protein nitration may contribute to underlying mechanism for the age-related functional decrement (Thompson and Brown 1999). 101Age-Related Decline in Actomyosin Structure and Function The results of this proteomic study revealed five modified proteins, identified by MALDI-TOF Mass Spectrometry and confirmed with MS/MS and Western immunoblotting included the sarcoplasmic reticulum Ca +2 -ATPase (SERCA2a), aconitase, b-enolase, TPI, and carbonic anhydrase III, exhibited an age-dependent increase in 3-NT content in both type I and type II muscles. Confirming the aging phenotype between the two different muscles, significant levels of 3-NT modifica- tion were present at an earlier age in the semimembranosus muscle. The biological function of the identified proteins include energy production (TPI, b enolase, aconitase, carbonic anhydrase III), and calcium homeostasis (SR Ca-ATPase). Previous studies reveal that mitochondrial aconitase is one of the major intracellular targets of nitric oxide, and the decrease in aconitase activity has been attributed to the direct reactions of nitric oxide with the iron-sulfur cluster (Patel et al. 2003). In addition, previous studies demonstrate oxidative modifica- tions of carbonic andydrase III in vivo with a concomitant decrease in catalytic activities in liver tissue. There is increasing evidence that links b-enolase and TPI as targets for nitration in Alzheimer’s disease. Taken together, these studies provide some insights about the molecular mechanisms (disturbance in energy metabolism) responsible for the observed phenotypic changes in skeletal muscle. 7.5 Carbonylation One prominent marker of oxidative stress in aging skeletal muscle is protein carbo- nylation. Protein carbonylation can occur through metal catalyzed oxidation. In this reaction metals (copper and iron) catalyze the formation of highly-reactive, short- lived hydroxyl radicals that modify nearby amino acids (e.g. proline, arginine, lysine, and threonine). Protein carbonylation can also occur through a reaction of nucleophilic amino acid side chains with lipid oxidation products (e.g., HNE). In this reaction lipid peroxidation leads to the generation of aldehyde-containing byproducts, which covalently modify nucleophilic amino acid side chains on proteins (cysteine, histidine and lysine). There are several ways to identify carbonylated proteins including (1) immuno- assays that are based on derivatization with 2,4-dinitrophenyhydrazine followed by treatment with anti-2,4-dinitrophenol antibodies and secondary peroxidase-labeled antibodies, and (2) biotin hydrazide for derivatization of proteins with carbonyl groups followed by advanced proteomic tools such as two-dimensional gel separa- tion and detection with fluorescently labeled avidin, affinity enrichment with biotin–streptavidin liquid chromatography tandem mass spectrometric (LC-MS/ MS) analysis, enrichment using avidin affinity chromatography, followed by LC-MS/MS, and enrichment using avidin affinity chromatography followed by iTRAQ-based quantitative proteomics (Fig. 13). Using enrichment protocols fol- lowed by advanced proteomic technology allows for the identification of proteins susceptible to carbonylation. 102 L.V. Thompson 7.6 Carbonylation: Identification of Susceptible Mitochondrial Proteins in Fast-Twitch and Slow-Twitch Muscle with Aging Differences in mitochondrial protein carbonylation may contribute to the age-related changes in muscle phenotype (fast- versus slow-twitch) described earlier in this chapter. Advanced quantitative proteomic profiling to identify proteins susceptible to carbonylation in a muscle type (slow- vs fast-twitch) and age-dependent manner yields very interesting results. With aging, fast-twitch muscle has twice as many carbonylated mitochondrial proteins compared to slow-twitch muscle (78 and 38 carbonylated proteins in the fast-twitch and slow-twitch muscle, respectively; Feng et al. 2008). Bioinformatic analysis of the set of carbonylated proteins, using Ingenuity Pathway Analysis (IPA) to identify functions and canonical pathways, reveals that the carbonylated proteins belong to pathways and functional classes already known to be impaired in aging skeletal muscle. IPA is a knowledge database generated from peer-reviewed scientific publications that enables discovery of highly repre- sented functions and pathways from large, quantitative data sets. Eight canonical pathways and six biological functions are common to both muscle types (Table 1). The carbonylated proteins unique to fast-twitch muscle map to two distinct pathway (cellular function/maintenance and cell death) and two distinct functions (tryptophan metabolism and synthesis/degradation of ketone bodies) in the IPA environment. In contrast, no significant functions or pathways are assigned to the carbonylated Fig. 13 Enrichment Strategy for the Identification of Carbonylated Proteins. In this strategy the carbonylated proteins are labeled with biotin hydrazide (derivatization of proteins with carbonyl groups) followed by enrichment using avidin affinity chromatography, and ulitimately identified by mass spectrometry -C=O -CH 2 -NH-NH- H 2 N-NH - Complex protein mixture with carbonyl-containing protein Biotin Label carbonyl with biotin hydrazide Release bound proteins Digest isolated proteins to peptides Capture labeled peptide using avidin affinity column Biotin Biotin Avidin 103Age-Related Decline in Actomyosin Structure and Function proteins identified only in slow-twitch muscle. The finding of distinct pathways and functions in fast-twitch muscle is potentially significant, given the fact that fast-twitch muscle is known to show more rapid decline with age than slow-twitch muscle does. 7.7 Age-Dependent Protein Carbonylation and Impaired Biochemical Functions Using a two-pronged proteomic strategy, determining changes in carbonylated proteins and changes in protein abundance with age, 20 of the identified susceptible proteins in fast-twitch muscle show significant increases in carbonylation with age. Although it is beyond the scope of this chapter to discuss each protein in detail, several proteins are highlighted. Voltage-dependent anion channel (VDAC) protein and its binding partner ADP/ATP translocase protein show significant increases in carbonylation with aging and map to “Cellular function and maintenance” within the IPA environment. VDAC enables transport of ions, such as calcium ions (Ca 2+ ), across the inner-mitochondrial membrane, critical to mitochondrial function. Interestingly, impaired mitochondrial cycling of Ca 2+ is associated with aging skel- etal muscle. Thus, it is possible to hypothesize that increased carbonyl modification of these proteins critical to mitochondrial inner membrane transport may contribute to this impaired cellular function in aged fast-twitch muscle. IPA enables identification of biochemical pathways represented by proteins showing changes in carbonylation with age that may not be apparent via visual inspection of the list of proteins. There are 13 canonical pathways and 7 biological functions represented by the proteins that increase in carbonylation with age (Table 2). Although it is beyond the scope of this chapter to discuss each pathway and function, several pathways are highlighted below to demonstrate the valuable tool of IPA. For instance, proteins with enzymatic activity mapping to five of the steps in fatty acid metabolism show increased age-dependent carbonylation. The identification of Table 1 Ingenuity Pathway Analysis (IPA) pathways and functions significantly represented by carbonylated proteins Canonical pathway Function Both muscle types Oxidative phosphorylation Carbohydrate metabolism Mitochondrial dysfunction Cell signaling Butanoate metabolism Energy production Fatty acid metabolism Amino acid metabolism Valine, leucine, and isoleucine degradation Lipid metabolism Citric cycle Small molecule biochemistry Fatty acid elongation Pyruvate metabolism Unique to Fast-twitch muscle Tryptophan metabolism Cellular function and maintenance Synthesis and degradation of ketone bodies Cell death 104 L.V. Thompson proteins showing susceptibility to carbonylation within the fatty acid metabolism pathway is very interesting based on (1) lipid content is known to increase in aging skeletal muscle, and (2) aging skeletal muscle has a decreased ability to oxidize fatty acid for energy generation. Decreased fatty acid metabolism may increase the presence of toxic lipids within skeletal muscle tissue, leading to more carbonylation, setting up a feedback scenario by which carbonylation impairs function and leads to further lipid perioxidation and modification and dysfunction of these proteins. 7.8 Glycation and Aging Skeletal Muscle Protein glycation is another likely explanation for skeletal muscle dysfunction with age. Advanced glycation end products (AGEs) are a diverse class of post-transla- tional modifications stemming from reactive aldehyde reactions. Because of the highly diverse reaction pathways leading to AGE formation, AGEs with a variety of chemical structures have been identified. The accumulation of AGEs is associ- ated in the pathogenesis of many degenerative diseases because AGEs reduces their susceptibility to degradation. N є -(carboxymethyl)lysine (CML, a 1-carboxyalkyl group is attached to the epsilon amino group of a lysine residue) is the major AGE-product in vivo and is often used as a biomarker of damage and increased oxidative stress. CML is formed by either oxidative breakdown of Amadori products or via adduction of lipid aldehydes generated from peroxidation of membrane (Fig. 14a, b). CML-modified proteins, determined biochemically and immunohistochemically, have extracellular as well as intracellular deposition. They are found in plasma, renal tissues, and retinas of diabetic patients and renal failure patients (Misselwitz et al. 2002; Saxena et al. 1999; Uesugi et al. 2001; Dyer et al. 1993; McCance et al. 1993). The severity of the Table 2 Significant canonical pathways mapped to protein showing age-dependent quantitative changes by IPA in fast-twitch muscle Canonical pathway Function Oxidative Phosphorylation Carbohydrate metabolism Mitochondrial dysfunction Cell signaling Fatty acid metabolism Energy production Valine, leucine, and isoleucine degradation Amino acid metabolism Citric cycle Lipid metabolism Fatty acid elongation Small molecule biochemistry Pyruvate metabolism Cell death Tryptophan metabolism Synthesis and degradation of ketone bodies Propanoate metabolism B-alanine metabolism Lysine degradation Glutathione metabolism 105Age-Related Decline in Actomyosin Structure and Function tissue lesion (e.g., atherosclerosis) correlates with the tissue AGE concentration (Marx et al. 2004). With age, the concentration of CML in tissues increases significantly in cartilage and skin collagen (Verzijl et al. 2000). These findings suggest glycoxidation reactions and oxidative stress may be involved in the development of age-related deterioration of skeletal muscle function. Although the basal level of glycation in muscle protein is small (0.2 mmol/mol lysine) there is a tenfold increase in the percentage of individual fibers containing CML-modified proteins with age (Fig. 14d). There are two characteristic patterns of the CML-immunolabeling of individual muscle fibers (intracellular punctuate labeling and labeling at the fiber periphery (Fig. 14c) suggesting that there are targeted or susceptible proteins. Fig. 14 Glycation and Aging Skeletal Muscle (Snow et al. 2007), N -(carboxymethyl)lysine (CML, a 1-carboxyalkyl group is attached to the epsilon amino group of a lysine residue) is the major AGE-product in vivo and is often used as a biomarker of damage and increased oxidative stress. Panel A, B – CML is formed by either oxidative breakdown of Amadori products or via adduction of lipid aldehydes generated from peroxidation of membrane. Panel C – There are two characteristic patterns of the CML-immunolabeling of individual muscle fibers (intracellular punctuate labeling and labeling at the fiber periphery) in skeletal muscle from very old rats. Panel D – There is a tenfold increase in the percentage of individual fibers containing CML-modified proteins with age. Panel E – Using proteomic technology (mass spectrometry and bioinformatics) to identify the proteins susceptible to CML-modification, the CML-modified proteins are critical enzymes involved in energy production Glucose + amino group of proteins, lipids and nucleic acids Schiff’s Base Amadori product Nonenzymatic Glycation a b c d e Classic Rearrangement CML Lipid Peroxidation CML Protein Score % coverage Peptides VDAC 112 54 11 B-enolase 88 37 11 CK 177 39 15 Actin 109 32 10 CAIII 74 46 6 0 15 30 % AGE Positive YOVO . about the molecular mechanisms responsible for age-related alterations in calcium regulation in skeletal muscle. 7.4 Aging Skeletal Muscle Phenotype – Nitration and Skeletal Muscle Proteins One. oxidative stress and the skeletal muscle aging phenotype, comparison of damaged skeletal muscle proteins in two muscles, the soleus and semimembranosus, each composed of different skeletal muscle fiber. of permeabilized muscle fibers (Callahan et al. 2001). Age-related oxidative damage of myosin and actin are probably accumu- lating in muscle as a consequence of decreased muscle protein turnover