Deborah A. Ferrington

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.

DIASTEREOSELECTIVE REDUCTION OF PROTEIN-BOUND METHIONINE SULFOXIDE BY METHIONINE SULFOXIDE REDUCTASE

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D‐sulfoxide diastereomer of CaM‐bound L‐MetSO (L‐Met‐D‐SO). After exhaustive reduction by pMSR, the ratio of L‐Met‐D‐SO to L‐Met‐L‐SO decreased to about 1:25 for hydrogen peroxide‐oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.

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.

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.

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.

Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging

We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0-20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca(2+)-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca(2+)-ATPase and to its association with proteins.

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle.

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.