Diana J. Bigelow

Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle.

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.

The Oxidative Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase by Peroxynitrite

The oxidative inactivation of rabbit skeletal muscle Ca(2+)-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO-) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations ( < or = 0.2 mM) resulted in a decrease of Ca(2+)-ATPase activity primarily through oxidation of sulfhydryl groups. Most of this deactivation (ca.70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca(2+)-ATPase activity. However, as long as peroxynitrite concentrations were kept < or = 0.45 mM, the efficacy of DTT to reverse Ca(2+)-ATPase inactivation was enhanced for reaction mixtures which initially contained 5 mM glutathione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher peroxynitrite concentrations ( > or = 0.45 mM) resulted in Ca(2+)-ATPase inactivation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4 recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca(2+)-ATPase of SR is involved in cellular Ca(2+)-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca(2+)-ATPase might significantly contribute to the loss of Ca(2+)-homeostasis observed under biological conditions of oxidative stress.

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca-ATPase of rat skeletal muscle during aging: a peroxynitrite-mediated process?

The SR Ca‐ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca‐ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca‐ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the in vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue imolies an age‐associated increase in susceptibility to oxidation by this species.

Decreased plasma membrane calcium transport activity in aging brain

We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.

Altered Turnover of Calcium Regulatory Proteins of the Sarcoplasmic Reticulum in Aged Skeletal Muscle

We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 ± 0.4, 6.3 ± 1.3, and 8.3 ± 1.3 days, respectively. A half-life of 14.5 ± 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 ± 5% and 25 ± 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 ± 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.

Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca2+ transport ATPase of sarcoplasmic reticulum.

III. Probes of catalytic and Ca = + binding domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~25 A. Probes of the nucleotide site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 B. Fluorescein isothiocyanate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 C, TNP-nuclcotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 D. Pyridoxal phosohate probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 E. Probes of calcium transport sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 F. ]ntramolecular crosslinking sludics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Comparison of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN) as free radical initiators: a spin-trapping study

Spin trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and its hydrophobic analogue 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO) was used to identify and to monitor the concentration of participating radical species in oxidation reactions initiated by azo compounds: water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and lipophilic 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN). Incubation of AAPH with spin traps in aqueous media produced alkoxyl radical spin adducts with hyperfine splitting constants being aN= 14.62 G, βaH= 15.29 G, γaH= 0.72 G and aN= 13.46 G, βaH= 12.53 G, for DMPO and DMPIO, respectively. In contrast, formation of AMVN-derived peroxyl radicals was detected by both direct EPR and spin trapping in DMSO (dimethylsulfoxide) solutions. In the presence of either rabbit skeletal sarcoplasmic reticulum (SR) membranes (10 g dm–3 of SR protein) or egg phosphatidylcholine liposomes (10 g dm–3 of lipid) preloaded with AMVN no spin adduct formation was observed, for both DMPO and DMPIO spin traps, indicating that AMVN-derived radical species do not escape the lipid environment. Only a small portion of AAPH-derived alkoxyl radicals was trapped by DMPIO in the presence of SR membranes. Spectral characteristics of the DMPIO spin adduct indicate its location at the lipid–water interface. At the same time, there was virtually no effect of SR on the rate of formation and steady-state level of the DMPO-spin adduct formed in the aqueous phase. From these data we suggest that the bulky cytosolic domains of the SR Ca2+-ATPase protect the membrane surface from radicals generated in the bulk (aqueous) solvent. Other evidence also demonstrates different mechanisms for free radical formation by AAPH and AMVN azo-initiators.

Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase.

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca(2+)- and Mg(2+)-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl (ITC-TEMPO), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca(2+)-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a-PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca(2+)-transport function.

Activation of the sarcoplasmic reticulum Ca2+-ATPase induced by exercise

Prolonged exercise has been shown to cause disruption of intracellular calcium homeostasis in skeletal muscle, which is normally maintained by the sarcoplasmic reticulum (SR) Ca2+-ATPase. We have investigated the response of this enzyme to increased intracellular calcium levels by investigating the functional and physical characteristics of the SR Ca2+-ATPase and membrane lipids following 2 h of treadmill running and throughout a period of post-exercise recovery. The Ca2+-ATPase of SR membranes purified from exercised rats shows increases in enzymatic activity correlating with post-exercise recovery time. Corresponding increases in active Ca2+-ATPase pump units are observed, as measured by the concentration of phosphorylated enzyme intermediate formed from ATP. However, catalytic turnover rates of the Ca2+-ATPase are unchanged. Using spin-label electron paramagnetic resonance to assess both membrane fluidity and associations between individual Ca2+-ATPase polypeptide chains, we find no exercise-induced alterations in membrane dynamics which could explain the observed increases in Ca2+-ATPase activity. Nor do we find evidence for altered membrane purification as a result of exercise. We suggest that the cell responds to the challenge of increased cytosolic calcium levels by increasing the proportion of functional SR Ca2+-ATPase proteins in the membrane for the rapid restoration of calcium homeostasis.

Age-related chemical modification of the skeletal muscle sarcoplasmic reticulum Ca-ATPase of the rat

Much emphasis has been placed on the description of age-related changes in skeletal muscle physiology. The present paper summarizes the chemical characterization of age-related post-translational modifications of the rat skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase isoforms SERCA1 and SERCA2a obtained from 5- and 28-month-old male Fischer 344 rats. Whereas the SERCA1 isoform shows an age-dependent loss of Cys and Arg, the SERCA2a isoform displays a loss of Cys but also a significant accumulation of 3-nitrotyrosine. The in vitro exposure of SR vesicles particularly rich in SERCA1 (>90%) from 5-month-old rats to low levels of peroxyl radicals yielded SR vesicles with physical properties of the SR Ca-ATPase identical to those observed for the SR Ca-ATPase obtained from 28-month-old rats. The peroxyl radical-modified SR Ca-ATPase showed a loss of Cys and Arg but also of Ser and Met, indicating that peroxyl radicals, though a good model oxidant to generate aged SR vesicles, may not be the only oxidant responsible for the chemical modification of the SR Ca-ATPase in vivo. In fact, efficient thiol modification of the SERCA1 was also observed after the exposure to peroxynitrite. Peroxynitrite selectively nitrated the tyrosine residues of the SERCA2a isoform even in the presence of an excess of SERCA1. Thus, peroxynitrite may be responsible for the age-dependent modification of the SR Ca-ATPase in vivo.