Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC–electrospray-tandem MS: selective protein oxidation during biological aging

Abstract

The selective reversible S-glutathiolation of specific SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) cysteine residues represents a novel physiologic pathway of NO (nitric oxide)-dependent arterial smooth muscle relaxation [Adachi, Weisbrod, Pimentel, Ying, Sharov, Schöneich and Cohen (2004) Nat. Med. 10, 1200-1207]. This mechanism may be impaired through the irreversible oxidation of functionally important cysteine residues as a consequence of oxidative stress and aging. To establish whether in vivo aging and in vitro oxidation by peroxynitrite result in the loss of such functionally important cysteine residues of SERCA, we have developed and optimized a quantitative method to monitor the oxidation state of the individual SERCA cysteine residues using a maleimide-based fluorescence dye, TG1 (ThioGlo 1), as a label for cysteine residues that have not been altered by oxidation and are not involved in disulphide bridges. A high efficiency for TG1 labelling of such residues and the chemical structure of cysteine-TG1 adducts were validated by MS analysis of model peptides, model proteins and rat skeletal muscle SERCA1. Tryptic peptides containing 18 out of a total of 24 cysteine residues were identified by HPLC-ESI (electrospray ionization)-MS/MS (tandem MS). Two cysteine residues, at positions 344 and 349, were detected in the form of an internal disulphide bridge, and another 16 were found to be labelled with TG1. Using HPLC-ESI-MS, we quantitatively mapped peroxynitrite oxidation of eight cysteine residues (positions 364, 417, 420, 498, 525, 674, 675 and 938), some of which are involved in the control of SERCA activity. Biological aging resulted in the partial modification of cysteine residues 377, 498, 525, 561, 614, 636, 674, 675, 774 and 938. Neither peroxynitrite exposure nor biological aging affected the apparent SERCA1 ATP affinity. Our data show an age-dependent loss of cysteine residues (approx. 2.8 mol of cysteine/mol of SERCA1), which may be partially responsible for the age-dependent decrease in the specific Ca2+-ATPase activity (by 40%).

Comparative proteomic profiling reveals a role for Cisd2 in skeletal muscle aging

“…As TAs appear to be associated with myopathy, Serca located in the TA membrane has been suggested to be inactivated or functionally compromised (Boncompagni, Protasi & Franzini‐Armstrong, 2012). Consistent with the above, our current findings in mice, and previous findings using a rat model (Sharov, Dremina, Galeva, Williams & Schoneich, 2006), have both revealed that Serca activity decreases in an age‐dependent manner and this decrease in Serca activity is correlated inversely with increased oxidative modification, namely cysteine sulfonation and 3‐nitrotyrosine, which occurs as a consequence of oxidative stress and aging.…”

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

“…SR/ER Ca 2+ ATPase (SERCA) is another target of redox modifications. Oxidation of SERCA1 in skeletal muscle by peroxinitrite causes an attenuation of activity of the Ca 2+ pump (Sharov et al, 2006). On the mitochondrial side, a vast body of evidence supports the activation of the PTP by ROS in cardiac muscle (Brookes et al, 2004;Halestrap et al, 2004).…”

Nitroso–Redox Interactions in the Cardiovascular System

“…8 In the case of the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), for example, oxidation at multiple cysteine residues leads to loss of function. 9 …”