Labeling the Ca2+-ATPase of Skeletal Muscle Sarcoplasmic Reticulum with Maleimidylsalicylic Acid

Velasco-Guillén, Isabel; Guerrero, Julio Becerra; Gómez‐Fernández, Juan C.; Teruel, José A.

doi:10.1074/jbc.m001871200

Cited by 2 publications

(3 citation statements)

References 44 publications

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“…It is interesting that only Cys 318 of SERCA2a cross-linked to the maleimide probe attached to PLB. Numerous previous studies have detected labeling of multiple Cys residues (residues 344, 364, 377, 471, 498, 614, 636, 670, and 674) of the Ca 2ϩ -ATPase with different sulfhydryl-reacting compounds, including maleimides (39,40). To our knowledge, however, this is the first report of any covalent modification of Cys 318 of the Ca 2ϩ pump.…”

Section: Discussionmentioning

confidence: 47%

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

Jones

Cornea

Chen

2002

Journal of Biological Chemistry

187

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Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca2؉ -ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca 2؉ . To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for crosslinking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-Å-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca 2؉ or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca 2؉ -free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys 318 of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys 318 of SERCA2a suggests that PLB may interfere with Ca 2؉ activation of SERCA2a by a protein interaction occurring near transmembrane helix M4.Ca 2ϩ -ATPase activities or SERCA 1 enzymes are 100-kDa integral membrane proteins responsible for the active transport of Ca 2ϩ into the sarco(endo)plasmic reticulum (1). In heart, the predominant Ca 2ϩ -ATPase expressed is SERCA2a(1), which pumps Ca 2ϩ into the SR lumen, causing cardiac muscle relaxation (2). A unique property of cardiac muscle is the regulation of SERCA2a by PLB, a small, single span membrane protein (3, 4), which inhibits the Ca 2ϩ -ATPase by decreasing its apparent affinity for Ca 2ϩ (5). Phosphorylation of PLB at Ser 16 by PKA and at Thr 17 by Ca 2ϩ /calmodulin-dependent protein kinase relieves the inhibitory action of PLB on SERCA2a, giving an increase in the rate of cardiac muscle relaxation as well as a positive inotropic effect (6, 7). Currently, there is much interest in understanding the molecular mechanism of SERCA2a inhibition by PLB, both as a paradigm for understanding membrane protein interactions and for the potential of targeting drugs to this system to treat heart failure (6, 7).Phospholamban has an interesting structure (6). Containing only 52 amino acids, the protein is a homopentamer in the membrane held together by Leu/Ile zipper interactions occurring in the transmembrane region (residues 32-52) (8). The cytoplasmic domain (residues 1-31) is highly charged and basic and is postulated to interact with SERCA2a by both electrostatic and hydrophobic interactions (9, 10). PLB mutagenesis studies suggest that the PLB monomer is the active inhibitory speci...

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Section: Discussionmentioning

confidence: 47%

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

Jones

Cornea

Chen

2002

Journal of Biological Chemistry

187

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“…The redox state of specific cysteine residues of SERCA is important for enzymatic function so that modifications of different SERCA cysteine residues may result in both inhibition and activation of the protein [13,14,31]. Earlier studies on the role of cysteine modifications in the regulation of SERCA activity were based on either selective labelling or site-directed mutagenesis of cysteine residues that resulted in a significant change in protein activity [13,[31][32][33][34][35][36]. Although these approaches specifically addressed SERCA modifications of selected cysteine residues, they are not so relevant to oxidative modifications of the protein cysteine residues in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Although these approaches specifically addressed SERCA modifications of selected cysteine residues, they are not so relevant to oxidative modifications of the protein cysteine residues in vivo. Previous studies involving proteolytic mapping of SERCA and HPLC-MS analysis detected several specific cysteine-containing peptides with a mass increase fitting to sulphoxidation, nitrosation and S-glutathiolation of SERCA cysteine residues, and quantified the loss of some cysteine residues upon oxidation in vitro using reactive thiol labelling [13,14,33]. However, all previous studies suffered from both incomplete cysteine labelling in SERCA and the use of peptide masses only (MS1 mode), but not MS/MS, for the identification of labelled peptides.…”

Section: Discussionmentioning

confidence: 99%

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

Sharov

Dremina

Galeva

et al. 2006

Biochemical Journal

100

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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%).

show abstract

Labeling the Ca2+-ATPase of Skeletal Muscle Sarcoplasmic Reticulum with Maleimidylsalicylic Acid

Cited by 2 publications

References 44 publications

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

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

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