J Muscle Res Cell Motil (2008) 29:119–126 DOI 10.1007/s10974-008-9145-x
ORIGINAL PAPER
Myosin as a potential redox-sensor: an in vitro study Chiara Passarelli Æ Stefania Petrini Æ Anna Pastore Æ Valentina Bonetto Æ Patrizio Sale Æ Laura M. Gaeta Æ Giulia Tozzi Æ Enrico Bertini Æ Monica Canepari Æ Rosetta Rossi Æ Fiorella Piemonte
Received: 25 June 2008 / Accepted: 28 August 2008 / Published online: 9 September 2008 Ó Springer Science+Business Media B.V. 2008
Abstract A balanced redox status is necessary to optimize force production in contractile apparatus, where free radicals generated by skeletal muscle are involved in some basic physiological processes like excitation–contraction coupling. Protein glutathionylation has a key role in redox regulation of proteins and signal transduction. Here we show that myosin is sensitive to in vitro glutathionylation and MALDI-TOF analysis identified three potential sites of glutathione binding, two of them locating on the myosin head. Glutathionylation of myosin has an important impact on the protein structure, as documented by the lower
fluorescence quantum yield of glutathionylated myosin and its increased susceptibility to the proteolytic cleavage. Myosin function is also sensitive to glutathionylation, which modulates its ATPase activity depending on GSSG redox balance. Thus, like the phosphorylation/dephosphorylation cycle, glutathionylation may represent a mechanism by which glutathione modulates sarcomere functions depending on the tissue redox state, and myosin may constitute a muscle redox-sensor.
ChiaraPassarelli and Stefania Petrini contributed equally to the work reported here.
Abbreviations GSH Reduced glutathione GSSG Oxidized glutathione GSPro Glutathionylated proteins ROS Reactive oxygen species
C. Passarelli S. Petrini L. M. Gaeta G. Tozzi E. Bertini F. Piemonte (&) Molecular Medicine Unit, Children’s Hospital and Research Institute ‘‘Bambino Gesu`’’, P.za S. Onofrio, 4, Rome 00165, Italy e-mail:
[email protected] C. Passarelli Department of Biology, University of Rome ‘‘Roma Tre’’, Rome, Italy A. Pastore Laboratory of Biochemistry, Children’s Hospital and Research Institute ‘‘Bambino Gesu`’’, Rome, Italy V. Bonetto ‘‘Mario Negri’’ Institute for Pharmacological Research, Dulbecco Telethon Institute, Milan, Italy P. Sale IRCCS San Raffaele La Pisana, Rome, Italy M. Canepari R. Rossi Institute of Human Physiology, University of Pavia, Pavia, Italy
Keywords Glutathionylation Oxidative stress Myosin Sarcomere
Introduction Protein glutathionylation is a dynamic process that is currently considered a mechanism of redox-mediated signal transduction, as well as a way for cells to store glutathione during oxidative stress and/or to protect critical protein cysteines from the irreversible oxidation, thus preventing permanent loss of function as a consequence of oxidative insult (Cotgreave and Gerdes 1998; Klatt and Lamas 2000; Fratelli et al. 2002; Giustarini et al. 2004; Fratelli et al. 2005). However, glutathionylated proteins have also been observed under basal conditions, suggesting their involvement in physiological signalling besides redox function regulation (Fratelli et al. 2002; Lind et al. 2002). Recently,
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we demonstrated the presence of constitutively glutathionylated proteins in human central nervous system and in fibroblasts, which increased after inducing a local oxidative stress (Pastore et al. 2003; Sparaco et al. 2006). Muscle function can be modulated by the cellular redox state, and ROS may be an important regulatory mechanism for normal contractile muscle apparatus. It has been suggested that some redox-sensitive thiols in the contractile filaments may represent putative ‘‘sensors’’ for changes in intracellular ROS levels by activating redox-sensitive signalling pathways (Andrade et al. 1998; Lamb and Posterino 2003). However, the identity of these ‘‘redox sensors’’ is not yet determined, although critical reactive cysteines are found in many key cardiac proteins including those implicated in ATP production, ionic homeostasis, signal transduction, transcription and excitation–contraction coupling (Shattock and Matsuura 1993; Ward et al. 1998; Choi et al. 2001; Eu et al. 2000). Contractile function in skeletal muscle fibres is sensitive to changes in cellular redox balance, with brief exposure to oxidants increasing force, and prolonged exposures decreasing it, thus indicating that the presumed cellular targets have optimal redox states (Andrade et al. 1998; Lamb and Posterino 2003). Glutathione could take part in these effects of the oxidants on the properties of the contractile muscle apparatus, as suggested by the experiments performed on rat skeletal fibres by Lamb and Posterino (Lamb and Posterino 2003; Murphy et al. 2008), even if the molecular basis of this ‘‘GSH effect’’ are not yet elucidated. The reversible thiol modifications regulated by changes of the redox status of glutathione may be nanoswitches to turn on and off proteins, similar to phosphorylation, in cells. Thus, in light of accumulating evidence indicating a crucial role for glutathione in the regulation of cellular signalling in response to oxidative and nitrosative stress and given that glutathionylation of proteins could have profound implications for cardiac function, either in physiological and pathological conditions, we analysed the in vitro glutathionylation of cardiac myosin and its effect on protein structure and functionality.
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Roche Diagnostics (Penzberg, Germany). All other chemicals were from Sigma-Aldrich (St. Louis, MI, USA). Heart samples were frozen in liquid nitrogen for biochemical study, and in liquid nitrogen-cooled isopentane for immunofluorescence analysis. Human hearts were obtained at autopsy 2–10 h after death, with the consent of the Institutional Ethical Committee. Pig myosin was obtained from Sigma-Aldrich (St. Louis, MI, USA). Myosin extraction from rat ventricular myocardium Rats (n = 5) were killed by ether anaesthesia and hearts were dissected out. Myosin was extracted according to Canepari et al. (2000). Briefly, the cardiac sample was put in a buffer (skinning solution) containing: 150 mM potassium propionate, 5 mM magnesium acetate, 5 mM sodium ATP, 5 mM EGTA, and 5 mM KH2PO4, and cut in small pieces. Myosin was extracted within 40 min in a buffer containing 0.4 M KCl, 0.1 M KH2PO4, 0.05 M K2HPO4, 0.01 M Na4P2O7, 4 mM MgCl2, 0.5 mM NaN3, 5 mM EDTA, 2 mM ATP, 2 mM DTT and protease inhibitor cocktail (complete, EDTA-free) pH 6.5. After 30 min centrifugation at 7,000 rpm, the supernatant was diluted in 18 volumes of 5 mM EDTA, 1 mM DTT and protease inhibitor cocktail solution, pH 6.6. After settling for 3 h, the precipitated myosin was collected by 30 min centrifugation at 7,000 rpm, and the pellet re-suspended in a buffer (myosin buffer 2X) containing: 0.05 M MOPS, 1.2 M KCl, 4 mM MgCl2, 2 mM EGTA, pH 7. Rat myosin was used for the activity measurements where we need freshly purified protein (within 5 days from the purification). Analysis of glutathione-conjugates by Western blot About 10 lg of pure rat cardiac myosin were applied onto 8% non-reducing SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane overnight at 70 mA. The membrane was blocked with 5% non-fat dry milk in TBST (100 mM NaCl and 10 mM Tris-HCl, pH 7.8 containing 0.1% Tween 20) for 2 h, at room temperature, and probed with anti-GSPro antibody (1/250) and anti-myosin antibody (1/250).
Materials and methods
Effect of GSSG on myosin thiols and glutathionylation
Chemicals
Glutathionylation of rat cardiac myosin (10 lg) was assessed by Western blot using an anti-GSPro antibody after incubation with various concentrations (0–10 mM) of GSSG for 10 min at 37°C. Changes in myosin thiols were determined by labelling the protein for 1 h at 37°C with 0.4 mM (final concentration) of (?)-biotinyl-3maleimidopropionamidyl-3,6-dioxa-octanediamine (maleimide PEO2-biotin). Western blot analysis of biotinylated
Mouse anti-GSPro was obtained from Virogen (Watertown, MA). Mouse anti-slow myosin was from Ylem (Italy). (?)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine (maleimide PEO2-biotin), streptavidin and BCA-protein assay were from Pierce (Rockford, IL). Protease inhibitor cocktail (complete, EDTA-free) was from
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proteins was subsequently performed using streptavidin (1:1,000).
1925) and expressed as lmol/mg protein per minute. The protein content was determined by BCA-protein assay.
Quantification of glutathionylated myosin by densitometry analysis
Identification of the glutathionylation sites of myosin
The extent of glutathionylation was quantified by analysing nitrocellulose filters on the Bio-Rad Model GS-670 imaging densitometer (Bio-Rad Laboratories, Hercules, CA). Data were analysed by the Bio-Rad Molecular AnalistTM Software, version 1.3 and normalized with the quantity of protein loaded on the gels. HPLC determination of glutathione bound to myosin About 6.8 mg/ml rat cardiac myosin was incubated for 2 h at room temperature with 5 mM (final concentration) GSSG. To 50 ll of glutathionylated myosin were added 25 ll of 12% sulfosalicylic acid; the protein pellet was then dissolved in 75 ll of 0.1 N NaOH and protein bound glutathione (GS-Pro) determined. The derivatization and chromatography procedures were performed, as previously reported (Pastore et al. 2003). Intrinsic fluorescence of glutathionylated myosin Intrinsic fluorescence measurements of native and 10 mM GSSG-treated rat cardiac myosin (1 h at 37°C) were performed on a Perkin Elmer LS 50 B spectrofluorimeter. The slit-width was fixed at 5 nm for excitation and emission. The samples were excited at 278 nm, and the emission was monitored between 300 and 400 nm.
About 1 mg/ml pure myosin was incubated for 2 h at room temperature with 5 mM (final concentration) GSSG according to previous reports (Casagrande et al. 2002; Ghezzi et al. 2006). Iodoacetamide was added to the reaction mixture to a final concentration of 5 mM to block residual free Cys residues. Control or glutathionylated myosin was digested under non-reducing conditions with trypsin (at a trypsin:myosin ratio of 1:10, wt/wt) in a solution containing 80% of acetonitrile and 50 mM ammonium bicarbonate for 10 min at 37°C (Casagrande et al. 2002; Ghezzi et al. 2006). Tryptic digests were concentrated and desalted using ZipTipÒ pipette tips with C18 resin and 0.2 ll bed volume (Millipore). Peptide-mass fingerprint analysis was carried out on a ReflexIIITM MALDI-TOF mass spectrometer (Bruker Daltonics) using a-cyano-4-hydroxycinnamic acid as matrix, and the mass spectra were internally calibrated with trypsin autolysis fragments, as previously described (Casoni et al. 2005). Database (SwissProt 47.3) searches were done using the Mascot program (http://www.matrixscience. com), allowing up to one missed trypsin cleavage and a mass tolerance of ±0.1 Da. In Mascot, searching all sequences deposited in SwissProt 47.3, scores greater than 66 were considered significant (P \ 0.05). Data were compared with the theoretical molecular weight of the myosin tryptic peptides, taking into consideration the possible glutathionylated Cys (?305.07 Da).
Limited proteolysis of glutathionylated myosin Results Pure rat cardiac myosin (30 lg) was incubated with or without 10 mM (final concentration) GSSG for 1 h at 37°C, and treated with various concentrations of trypsin for 15 min at 4°C. Samples were loaded on SDS 8% electrophoresis and stained with Coomassie brilliant blue. Determination of myosin ATPase activity assay in presence of increased concentrations of GSSG Ca-stimulated ATPase activity of rat cardiac myosin pretreated with increased GSSG concentrations (0–10 mM) for 2 h at 37°C was assayed in a medium containing 0.3 M KCl, 50 mM Tris-HCl (pH 7.6), 10 mM CaCl2, 2 mM Na2ATP at 27°C (Cappelli et al. 1989). The reaction was started by adding 50 lg of myosin, carried out for 5 min and stopped by adding 10% trichloroacetic acid. The amount of inorganic phosphate released was determined with the Fiske–Subbarow method (Fiske and Subbarow
We studied myosin glutathionylation by in vitro incubating the purified protein with increasing concentrations of GSSG (Fig. 1). Western blot analysis using a monoclonal anti-GSPro antibody, which specifically reacts with glutathione bound to proteins showed a dose-dependent increase in glutathionylated myosin (Fig. 1b) with a corresponding decrease in myosin-free thiols [as measured by Western blot analysis after maleimide PEO2-biotin labelling (Fig. 1a)]. These results indicate that thiols in myosin are lost in a dose-dependent manner upon GSSG treatment. The glutathionylation extent of myosin, as assayed by HPLC analysis, was 0.159 mol GSH/mol myosin for nontreated myosin and 0.954 mol GSH/mol myosin for GSSGtreated myosin (data not shown). The increase of protein glutathionylation was completely reversed by the subsequent addition of 0.5 mM DTT for 1 h at 37°C (data not shown).
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Fig. 1 Effect of GSSG on myosin protein thiols. (a) Western blot analysis after maleimide PEO2-biotin labelling showing the loss of myosin protein thiols following treatment with GSSG for 10 min at 37°C, and the corresponding semiquantitative analysis (densitometry). (b) Western blot showing glutathionylation of myosin
Fig. 2 Structural changes in glutathionylated myosin. Steady-state emission spectra of intrinsic fluorescence of native (upper trace), myosin treated with (lower trace) and without (middle trace) 10 mM GSSG for 1 h at 37°C were analysed in a spectrofluorimeter. Spectra were obtained using an excitation wavelength of 278 nm and excitation/emission slits of 5 nm
Then, in an attempt to evaluate the effects of glutathionylation on the conformation of myosin, the intrinsic fluorescence of the aromatic amino acids in each of the various forms of the protein was measured. Upon excitation of native myosin at 278 nm, an emission spectrum with a maximum at 346 was obtained (Fig. 2). The fluorescence spectra of native (upper trace) and GSSG-treated myosin (lower trace), normalized to the protein content, showed that glutathionylated myosin displays a decrease in the quantum yield of the emission spectra, thus showing the modification of the conformational integrity in glutathionylated myosin. Also the susceptibility of the glutathionylated myosin to trypsin proteolysis appeared increased compared to the native protein (Fig. 3). Presumably, the structural changes following glutathionylation lead myosin to be more sensitive to the attack by proteases. This is confirmed when
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determined by anti-GSPro antibody, and the corresponding semiquantitative analysis (densitometry). The blots are representative of one of three independent experiments for each condition tested. For details see ‘‘Materials and Methods’’
Fig. 3 Effect of glutathionylation on the proteolytic digestion of purified myosin. (a) Native and 10 mM (final concentration) GSSGtreated myosin were incubated with various concentrations of trypsin for 15 min at 4°C. Samples were separated by SDS-PAGE, and protein bands were visualized by Coomassie staining. The gel is representative of one of three independent experiments. For details see ‘‘Materials and Methods’’. (b) Densitometry analysis of the proteolytic bands
myosin was treated with GSSG, subjected to tryptic digestion under non-reducing conditions and the peptide mass fingerprint analysed by MALDI-TOF MS. Interestingly, glutathionylated myosin (Fig. 4b) was more susceptible to tryptic digestion than the unmodified protein (Fig. 4a), as judged by the higher number and largely more intense tryptic fragment ions detected in the mass spectra of the GSSG-oxidized protein. In addition, web based screening of protein database indicated, with a significant score (139 Mascot score), that the peptide mass fingerprint of the
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Fig. 4 Glutathionylated myosin is more susceptible to trypsin digestion than unmodified myosin. Representative MALDI-TOF mass spectra of peptide mass fingerprints of cardiac myosin (a), in vitro glutathionylated cardiac myosin (b), and a spectrum zoom of the S-glutathionylated fragment 1,265.66 m/z (c). Tryptic digestion was performed under non-reducing conditions starting from the same myosin stock solution following treatment (b) or not (a) with 5 mM of GSSG, as described in Materials and Methods
GSSG-oxidized protein matched in large part (43 matched peptides, 25% sequence coverage) with the theoretical peptide mass fingerprint of myosin heavy chain, cardiac muscle beta isoform (SwissProt, accession number P79293). In the mass spectra of glutathionylated myosin, three peaks, not present in the spectra of the unmodified protein tryptic digest, at 1,265.66 (Fig. 4c), 1,626.77 and 2,464.31 m/z were detected, which matched with the masses of glutathionylated fragments 695–703, 942–952 or 941–951 and 384–403 (Table 1). This indicated that Cys400, Cys695 and Cys947 are potential glutathionylation sites of the myosin heavy chain, cardiac muscle beta isoform.
Finally, in order to evaluate if glutathionylation could interfere with cardiac myosin activity, Ca-stimulated ATPase activity was determined in presence of increasing GSSG concentrations. As reported in Fig. 5, incubation with (0– 10 mM) GSSG resulted in a dose-dependent response, with a mild increase (13%) of the enzymatic activity at low GSSG concentrations (up to 0.1 mM GSSG) and a progressive decrease, that reached 47% of inhibition, at higher GSSG concentration (10 mM). These findings lead us to hypothesize the presence of a multiple sulfhydryl groups in myosin, with different sensitivities to redox probes and opposed functional effects under an oxidative insult.
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Table 1 MALDI-TOF analysis of the peptide mass fingerprint of GSSG-treated cardiac myosin: potential glutathionylated sites m/z expa
m/z calb
Tryptic fragment sequence
GS-Cysc
1,265.66
1,265.56 (960.49 ? 305.07)
695
695
1,626.77
1,626.67 (1,321.60 ? 305.07)
942
947
1,626.77
1,626.67 (1,321.60 ? 305.07)
941
2,464.18 (2,159 ? 305.07)
384
2,464.33 a
CNGVLEGIR703 NVEDECSELKR952 RNVEDECSELK951 SAYLMGLNSADLLKGLCHPR
947 403
400
Experimental value
b
Calculated value considering the addition of a glutathione moiety
c
Glutathionylated Cys
Fig. 5 Effect of increasing concentrations of GSSG on cardiac myosin Ca-stimulated ATPase activity. (a) 50 lg cardiac myosin were incubated (10 min at 37°C) with indicated amounts of GSSG and then assayed for activity as described under Materials and Methods. Values are means ± S.D. (n = 3) and are expressed as mmol/min/mg protein. (b) Western blot showing glutathionylation of myosin determined by anti-GSPro antibody, as described in Materials and Methods, and the corresponding semiquantitative analysis (densitometry, (c)). The blots are representative of one of three independent experiments
Discussion Glutathione provides a means of regulating protein function by a mechanism, called glutathionylation, where the protein thiol groups are reversibly bound to glutathione (Klatt and Lamas 2000; Giustarini et al. 2004).
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Several proteins, participating in translation, molecular chaperoning, glycolysis, cell growth, cytoskeletal structure, antioxidant activity and signal transduction, are redoxsensitive (Lin et al. 2002; Cumming et al. 2004), and the reversible modifications of their cysteines may have a dual role of protection from irreversible oxidation and modulation of protein function by redox regulation. Therefore, protein glutathionylation can be considered a normal physiological process, but it can be greatly increased by oxidative stress, and accumulation of glutathionylated proteins has been reported in different cell types (for review, see reference. Klatt and Lamas 2000; Fratelli et al. 2004). Some of the proteins found to be glutathionylated belong to the class of cytoskeletal proteins. In particular, the glutathionylation of actin has been observed in several cells under varying conditions of oxidative stress, e.g. gastric mucosal cells treated with H2O2 or diamide, phorbol myristate acetate stimulated murine macrophages and human neutrophils (Chai et al. 1994; Rokutan et al. 1994). H2O2 treatment is also responsible for actin glutathionylation in human epidermal carcinoma A431 cells, where it regulates actin polymerisation (Wang et al. 2001). Under physiological conditions, protein glutathionylation may represent an antioxidant device that reduces the impact of oxidative stress, but at the same time can modulate protein activity if critical protein thiols are involved. Glutathionylation should also determine whether cells undergo apoptosis or necrosis, with low level of glutathionylation appeared to be associated with the apoptotic response, while high and sustained levels were associated with necrosis (Di Stefano et al. 2006). Thus, protein glutathionylation may represent an important metabolic answer to cellular redox regulation, with ROS acting as second messengers within several signal pathways (Cumming et al. 2004; Eaton et al. 2002; Xie et al. 1999; Brennan et al. 2004). ROS are implicated in many diseases, and play an important role in mediating cardiac hypertrophy (Sawyer et al. 2002; Amin et al. 2001), and during ischaemiareperfusion (Eaton et al. 2002; Berlett and Stadtman 1997; Bolli et al. 1988). Even excitation–contraction coupling of
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cardiac muscle seems to be very sensitive to shifts in redox balance, suggesting that contractile function of heart may depend on the redox state of some participating proteins (Krenz et al. 2003). A low, basal level of protein S-thiolation has been reported in the perfused isolated rat heart, with cytosolic, myofilament and cytoskeletal proteins as major substrates during the pro-oxidizing condition in myocardium (Eaton et al. 2002). In this study we analyse the in vitro glutathionylation of pure cardiac myosin and its effect on protein structure and functionality. Some critical Cys residues have been previously identified on rabbit myosin polypeptide, and modification of Cys-707 has been shown to affect ATPase activity, by inducing changes in the affinity for ADP (Konno et al. 2000). Glutathionylated myosin, under basal conditions, has been found in human T lymphocytes (Fratelli et al. 2002) and in human hepatoma cells (Fratelli et al. 2003), but its functional significance is still unclear. The presence of redox-sensitive free thiols on myosin has been evidenced when we in vitro glutathionylated myosin by incubating the purified protein with increasing concentrations of GSSG. We obtained a dose-dependent increase of glutathionylation with a corresponding decrease in myosin-free thiols. Furthermore, myosin glutathionylation is completely reversed by the treatment with a reducing agent, demonstrating that GSSG acts by modifying some susceptible cysteines on the protein through the formation of mixed disulphides. Moreover, we found that the glutathionylated myosin is more easily digested by trypsin compared to the untreated protein, possibly due to increasing exposure of residues normally hidden in the protein structure. This confirms that glutathionylation has an important impact on the structure of the protein, as demonstrated by the lower fluorescence quantum yield of glutathionylated myosin respect to the native form, and by the higher sensitivity to the proteolytic digestion. In order to identify the exposed cysteine residues that undergo glutathionylation, we analysed in vitro glutathionylated purified myosin by MALDI-TOF MS. The analysis showed three potential sites of glutathione binding (Cys 400, Cys 695 and Cys 947). Interestingly, two of these cysteine residues, Cys400 and Cys695, are located on the myosin head. The ATPase activity of cardiac myosin, measured in presence of increasing GSSG concentrations, showed a dose-dependent response which seems to suggest the existence of a redox-threshold of myosin activity. Andrade et al. (1998) previously demonstrated that the cellular redox balance in resting fibres is in relatively reduced state and that this is not optimal for contractile function. They showed that a brief exposure to H2O2 bring the cellular redox balance to the oxidation range and
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increased force. As exposure to H2O2 continued, the redox balance moved further into the oxidation state, and force decreased. A similar behaviour probably occurs in our conditions by using GSSG as pro-oxidant, where a range of GSSG concentrations around 0.1 mM stimulated ATPase activity of myosin, whereas levels higher than 1 mM significantly decreased it. However, such as in the model of Andrade et al. (1998) a prolonged oxidant exposure led to an oxidation state that decreased the force, similarly in our hands a drastic treatment with 10 mM GSSG switched to an inactivated form of myosin. The presence of some redox-sensitive thiols in the contractile filaments has been previously suggested (Andrade et al. 1998; Lamb and Posterino 2003). They could constitute putative ‘‘sensors’’ for changes in intracellular ROS levels by activating redox-sensitive signalling pathways. We propose that at least myosin may represent one of these redox sensors by changes of the redox status of glutathione, not excluding that some other redox sensors, upstream of those in myosin and with different sensitivity, could contribute to the redox signalling in the contractile apparatus. Therefore, despite phosphorylation/dephosphorylation, protein glutathionylation may represent a mechanism by which glutathione can modulate sarcomere functions depending on the redox state of the tissue. Acknowledgments This study has been supported by grants from ‘‘Fondazione Pierfranco e Luisa Mariani’’—Italy, Cariplo Foundation (to VB.) and Telethon Foundation-Italy (to VB., project n. S01010). VB. is an Assistant Telethon Scientist.
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