Heat-shock proteins attenuate SERCA inactivation by the anti-apoptotic protein Bcl-2: possible implications for the ER Ca2+-mediated apoptosis.

We have demonstrated previously that Bcl-2 and Bcl-2Δ21, a C-terminally truncated Bcl-2 sequence, inactivate SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) 1 in isolated SR (sarcoplasmic reticulum), accompanied by a translocation from CRDs (caveolae-related domains) of the SR. In the present study, we obtained evidence for the interaction of Bcl-2 with SERCA2b in C2C12 myoblasts and HEK (human embryonic kidney)-293 cells. Bcl-2 and SERCA2b co-immunoprecipitated from lysate and microsomal fractions of Bcl-2-overexpressing cells. However, Bcl-2 overexpression resulted only in a slight translocation from the CRDs and no significant SERCA inactivation. In isolated HEK-293 cell microsomes, incubation with Bcl-2Δ21 afforded SERCA2b inactivation and some translocation. HSP (heat-shock protein) 70, HSP90, HSP27 and α-crystallin attenuated Bcl-2Δ21-dependent SERCA2b inactivation. An in vitro mechanistic study with the SERCA1 isoform shows that HSP70 (i) protects SERCA1 from the inactivation by Bcl-2Δ21, (ii) inhibits SERCA1 translocation from CRD fractions, and (iii) prevents the Bcl-2Δ21-dependent loss of FITC labelling. Our data demonstrate that the mechanism of SERCA inactivation by Bcl-2 established in vitro for the SERCA1 isoform can be extended to the main housekeeping SERCA2b isoform, and that functional interactions of SERCA2b and Bcl-2 in the cell may be modulated by HSP70 and other chaperones and stress-regulated proteins.

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.

Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 µM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 µM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 µg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.

Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.

RATIONALE: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown. OBJECTIVES: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken. METHODS: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots. MEASUREMENTS AND MAIN RESULTS: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha. CONCLUSIONS: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite.

Selective fluorogenic derivatization of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine in peptides: a method designed for quantitative proteomic analysis.

There is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization of 3-nitrotyrosine (after reduction to 3-amino-tyrosine) and 3,4-dihydroxyphenylalanine with benzylamine derivatives to yield 6-amino- and 6-benzylamine-substituted benzoxazoles, which display characteristic fluorescence properties. The methodology can be expanded to other substituted benzylamines, which carry functional groups for affinity enrichment.

Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid peroxides.

The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys674 and Cys675, where Cys674 (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1. This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from dl-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein-derived peroxides as important mediators of oxidative stress in vivo, which may cause a selective oxidation of Cys residues leading to inactivation of membrane proteins.

Displacement of SERCA from SR lipid caveolae-related domains by Bcl-2: a possible mechanism for SERCA inactivation.

Bcl-2 exerts its anti-apoptotic effect in part through the regulation of Ca2+ homeostasis at the level of the endoplasmic reticulum. Earlier, we demonstrated that a truncated form of Bcl-2, Bcl-2delta21, interacts with and destabilizes the skeletal muscle sarco/endoplasmic reticulum Ca-ATPase (SERCA) [Dremina, E. S., Sharov, V. S., Kumar, K., Zaidi, A., Michaelis, E. K., and Schöneich, C. (2004) Biochem. J. 383, 361-370]. Here we show that (i) the transmembrane (TM) domain of Bcl-2 accelerates SERCA inactivation, (ii) both Bcl-2delta21 and full-length Bcl-2 selectively interact with SERCA1, and (iii) the inactivation of SERCA is accompanied by a translocation of SERCA from caveolae-related domains (CRD) of the sarcoplasmic reticulum (SR). In rat skeletal muscle SR, intact SERCA1 was detected only in the CRD fractions of a sucrose density gradient. Co-incubation of SR with either Bcl-2delta21 or full-length Bcl-2 resulted in both the appearance of Bcl-2delta21 or Bcl-2 in the fractions containing SERCA1 and translocation of SERCA1 from CRD fractions; the latter effect correlated with the loss of the Ca-ATPase activity of the protein.

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.

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%).

Protein tyrosine nitration in rat brain is associated with raft proteins, flotillin-1 and alpha-tubulin: effect of biological aging.

Protein 3-nitrotyrosine (3-NY) immunoreactivity of rat brain homogenate was localized to a ca. 50 kDa protein band by western blot (WB) analysis. The nitrated proteins were localized to the raft fraction obtained by centrifugation of the homogenate in a sucrose density gradient, which contained specific raft markers such as flotillin-1 and caveolin-1. Purification of the nitrated raft proteins either by a combination of reversed-phase high-performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or by immunoprecipitation (IP) with protein- and modification-specific antibodies coupled to WB and HPLC-electrospray ionization-tandem mass spectrometry (ESI--MS/MS) analysis allowed us to identify two proteins modified by 3-NY: flotillin-1 and alpha-tubulin. Both alpha- and beta-tubulin were detected in the rat brain raft fraction as abundant proteins, which co-immunoprecipitate with flotillin-1 and caveolin-1. Importantly, some protein-protein interactions in rafts were disrupted in 3-NY-containing proteins, e.g. caveolin-1 was dissociated from a complex with flotillin-1 and alpha-tubulin. The analysis of age dependencies did not show any significant change in protein nitration and expression of flotillin-1 and alpha-tubulin, but a decrease in the brain caveolin-1 level for old (34 months) versus young (6 months) rats. The putative mechanism of nitric oxide synthase (NOS) activity regulation by the level of caveolin expression and raft protein-protein interactions is discussed.

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.