In the present work we address the oxidative modifications accounting for the structural and functional impairment of the actomyosin complex under the oxidative stress mediated by peroxynitrite (ONOO-). Experiments on purified myosin and actin have shown that submicromolar ONOO- concentrations produce
strong inhibition of the F-actin stimulated myosin ATPase activity. The
peroxynitrite-induced actomyosin impairment correlated with structural modifications
that decrease the thermal stability of both actin and myosin leading to partially unfolded states. The results suggest a major role for the highly reactive cysteines on actin and on myosin and also for some critical methionines on G-actin. 3-nitrotyrosine does not contribute significantly to the observed functional alterations

Aureliano, M.

Gutiérrez-Merino, Carlos

Santos, Ana

Silva, D.

Tiago, Teresa

Universidade do Algarve

©2007 by MEDIMOND S.r.l. 187HX10C0093
Actomyosin Modulation by Peroxynitrite
T. Tiago1,2, D. Silva1, A. Santos1,
M. Aureliano1 and C. Gutiérrez-Merino2
1CCMar, Depto. de Química e Bioquímica, FCT,
Universidade do Algarve, 8000-139 Faro, Portugal;
2Depto. Bioquímica y Biologia Molecular, Facultad de Ciencias,
Universidad de Extremadura, 06071-Badajoz, Spain
Summary
In the present work we address the oxidative modifications accounting for
the structural and functional impairment of the actomyosin complex under the
oxidative stress mediated by peroxynitrite (ONOO-). Experiments on purified
myosin and actin have shown that submicromolar ONOO- concentrations produce
strong inhibition of the F-actin stimulated myosin ATPase activity. The
peroxynitrite-induced actomyosin impairment correlated with structural modi-
fications that decrease the thermal stability of both actin and myosin leading
to partially unfolded states. The results suggest a major role for the highly
reactive cysteines on actin and on myosin and also for some critical methionines
on G-actin. 3-nitrotyrosine does not contribute significantly to the observed
functional alterations.
Introduction
Peroxynitrite (PN) is increasingly recognized as playing a major role in the
skeletal and cardiac muscle dysfunction. This is supported by detection of
protein 3-nitrotyrosine (3-NT), a protein modification produced by the reac-
tion of PN with tyrosine, on skeletal and cardiac muscle proteins during aging
[1] or in diseases associated with myocardial inflammation or ischemia/reperfusion
insults [2,3]. Although these studies point to a correlation of protein nitration
with functional and structural modifications, the mechanism by which PN
may impair muscle contractility remains far from being elucidated. During
inflammation or ischemia/reperfusion episodes, tissue cells are exposed to
fluxes of PN ranging from submicromolar to micromolar concentrations [4].
Therefore, in order to mimic a chronic exposure to PN we have used 3-
188 European Meeting of the Society for Free Radical Research
morpholinosydnonimine (SIN-1), a compound that slowly decomposes in nitric
oxide (·NO) and superoxide anion (·O2-), which react each other to produce
ONOO-. In this work, we report the functional and structural impairment of
the myofibril proteins – myosin and actin - under the oxidative stress medi-
ated by PN. In addition, the relevance of different protein modifications com-
monly produced by PN to the actomyosin impairment is evaluated.
Materials and Methods
Myosin and actin were isolated from rabbit skeletal muscle and the F-actin
stimulated myosin ATPase activity measured spectrophotometrically using
the coupled enzyme pyruvate kinase/lactate dehydrogenase assay as in previ-
ous works [5,6]. Differential Scanning Calorimetry measurements were per-
formed on a Micro-Cal MC-2 operated at a scanning rate of 60ºC/h and under
a nitrogen pressure of 1.5-2 kg/cm2 during the scan. Titration of cysteines was
done with DTNB, as previously indicated [5,6]. Methionine oxidation was
assessed by SDS-PAGE densitometric analysis of the actin digestion products
produced by a-chymotrypsin and subtilisin as in Dalle-Donne et al. [7]. The
extent of 3-NT formation was assessed either spectrophotometrically or by
Western-blotting as in Tiago et al. [5,6].
Results and Discussion
In vitro exposure of actin to micromolar peroxynitrite fluxes promoted an im-
balance of the G-actin (monomer)/F-actin (filament) equilibrium which can effi-
ciently block the stimulation of the myosin ATPase activity by actin. Furthermore,
incubation of purified myosin with SIN-1 also resulted in inhibition of the myosin
ATPase activity stimulated by actin. The IC50 values, obtained from the nonlinear
least square fit of the data shown in Figure 1, take place at molar ratios of 6.12±0.86
and 7.351.53 mol SIN-1/mol of myosin and actin, respectively, showing that both
proteins are highly sensitive to PN oxidation. The loss of myofibril actomyosin
ATPase activity parallels a decrease of the thermal stability of both proteins. This
has been shown by the decrease of the unfolding enthalpy (DH) and critical tem-
perature (Tc) of SIN-1 treated myosin and G-actin in the DSC experiments (Ta-
ble 1). Moreover, the lack of significant decrease in the cooperative unit (DHV)
189Vilamoura, Algarve, Portugal, October 10-13, 2007
Figure 1. Inhibition of the myosin ATPase activity stimulated by actin upon SIN-1 treatment.
Activity assays were done with F-actin (squares) or myosin subfragment-1 (circles) treated
for 2h with different SIN-1 concentrations in 10mM Tris (pH 8.5), 25 mM KCl as indicated
in Materials and Methods.
Figure 2. Molecular model of G-actin (PDB Id: 1J6Z) on the left, and myosin subfragment-
1 (PDB Id: 2MYS) on the right, depicting the putative residues modified by PN. ATP and
Ca2+ represented as yellow and orange spheres, respectively, are shown just to indicate the
active site on the actin structure. Cyst and Met residues are represented in red and blue
stick-and-balls, respectively. The 25kDa tryptic fragment of myosin is coloured green where
it is indicated in purple the putative nitrated Tyr residues.
190 European Meeting of the Society for Free Radical Research
points out that PN is inducing partial unfolded states of myosin and G-actin which
can be fully prevented by incubating the sample with GSH during SIN-1 treatment.
To address the oxidative modifications accounting for the structural and functional
impairment of the actomyosin complex under the oxidative stress induced by PN,
different protein modifications commonly produced by PN were assessed as de-
scribed in Materials and Methods. The results suggested a major role for Cys-707
and Cys-697 on myosin, two highly reactive cysteines located close to the nucle-
otide binding site (Figure 2) whose modification has been previously shown to
have dramatic effects on the functional properties of myosin. 3-NT formation could
be detected in the 25 kDa tryptic myosin fragment (Figure 2), consistent with the
location of the four nitrated tyrosines in the myosin heavy chain extracted from
cardiac muscle of aged rats [8]. However, the extent of this chemical modifica-
tion is very low, i.e. less than 0.05 moles 3-NT/mole of myosin when 50% of the
activity is already lost. On actin, besides the highly reactive Cys-374, we found a
significant contribution of Met-44 and Met-47 to the observed impairment. While
Cys-374 is located at the C-terminal region which is the binding site for several
actin-binding proteins (including myosin), Met-44 and Met-47 are located in a loop
implicated in monomer/monomer contact (Figure 2). Therefore, the modification
of these critical residues by PN can directly affect the functional interaction with
myosin as well as the G-actin/F-actin equilibrium. 3-NT formation do not seem
to significantly contribute to the actomyosin ATPase activity impairment.
Acknowledgements
T. Tiago is supported by a post-doctoral grant (SFRH/BPD/20777/2004)
from the Portuguese Foundation for Science and Technology (FCT). Work
funded by Grant 3PR05A078 of Junta de Extremadura.
References
[1] J. KANSKI et al. Proteomic analysis of protein nitration in aging skeletal muscle
and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray
ionization tandem mass spectrometry, J Biol Chem, 280, 24261-24266 (2005).
[2] N.W. KOOY et al. Extensive tyrosine nitration in human myocardial inflamma-
tion: evidence for the presence of peroxynitrite, Crit Care Med, 25, 812-819
(1997).
[3] M.J. MIHM et al. Impaired myofibrillar energetics and oxidative injury during
human atrial fibrillation, Circulation, 104, 174-180 (2001).
[4] M.P. MURPHY et al. Peroxynitrite: a biologically significant oxidant, Gen Pharmacol,
31, 179-186 (1998).
[5] T. TIAGO et al. Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite,
Biochemistry, 45, 3794-3804 (2006).
[6] T. TIAGO et al. Peroxynitrite induces F-actin depolymerization and blockade of
myosin ATPase stimulation, Biochem Biophys Res Commun 342, 44-49 (2006).
[7] I. DALLE-DONNE et al. The tert-butyl hydroperoxide-induced oxidation of actin
Cys-374 is coupled with structural changes in distant regions of the protein, Bio-
chemistry, 38, 12471-12480 (1999).
[8] S.J. HONG et al. Proteomic analysis of age dependent nitration of rat cardiac
proteins by solution isoelectric focusing coupled to nanoHPLC tandem mass
spectrometry, Exp Gerontol, 42, 639-651 (2007).


Actomyosin modulation by peroxynitrite

Sapientia

©2007 by MEDIMOND S.r.l. 187HX10C0093Actomyosin Modulation by PeroxynitriteT. Tiago1,2, D. Silva1, A. Santos1,M. Aureliano1 and C. Gutiérrez-Merino21CCMar, Depto. de Química e Bioquímica, FCT,Universidade do Algarve, 8000-139 Faro, Portugal;2Depto. Bioquímica y Biologia Molecular, Facultad de Ciencias,Universidad de Extremadura, 06071-Badajoz, SpainSummaryIn the present work we address the oxidative modifications accounting forthe structural and functional impairment of the actomyosin complex under theoxidative stress mediated by peroxynitrite (ONOO-). Experiments on purifiedmyosin and actin have shown that submicromolar ONOO- concentrations producestrong inhibition of the F-actin stimulated myosin ATPase activity. Theperoxynitrite-induced actomyosin impairment correlated with structural modi-fications that decrease the thermal stability of both actin and myosin leadingto partially unfolded states. The results suggest a major role for the highlyreactive cysteines on actin and on myosin and also for some critical methionineson G-actin. 3-nitrotyrosine does not contribute significantly to the observedfunctional alterations.IntroductionPeroxynitrite (PN) is increasingly recognized as playing a major role in theskeletal and cardiac muscle dysfunction. This is supported by detection ofprotein 3-nitrotyrosine (3-NT), a protein modification produced by the reac-tion of PN with tyrosine, on skeletal and cardiac muscle proteins during aging[1] or in diseases associated with myocardial inflammation or ischemia/reperfusioninsults [2,3]. Although these studies point to a correlation of protein nitrationwith functional and structural modifications, the mechanism by which PNmay impair muscle contractility remains far from being elucidated. Duringinflammation or ischemia/reperfusion episodes, tissue cells are exposed tofluxes of PN ranging from submicromolar to micromolar concentrations [4].Therefore, in order to mimic a chronic exposure to PN we have used 3-188 European Meeting of the Society for Free Radical Researchmorpholinosydnonimine (SIN-1), a compound that slowly decomposes in nitricoxide (·NO) and superoxide anion (·O2-), which react each other to produceONOO-. In this work, we report the functional and structural impairment ofthe myofibril proteins – myosin and actin - under the oxidative stress medi-ated by PN. In addition, the relevance of different protein modifications com-monly produced by PN to the actomyosin impairment is evaluated.Materials and MethodsMyosin and actin were isolated from rabbit skeletal muscle and the F-actinstimulated myosin ATPase activity measured spectrophotometrically usingthe coupled enzyme pyruvate kinase/lactate dehydrogenase assay as in previ-ous works [5,6]. Differential Scanning Calorimetry measurements were per-formed on a Micro-Cal MC-2 operated at a scanning rate of 60ºC/h and undera nitrogen pressure of 1.5-2 kg/cm2 during the scan. Titration of cysteines wasdone with DTNB, as previously indicated [5,6]. Methionine oxidation wasassessed by SDS-PAGE densitometric analysis of the actin digestion productsproduced by a-chymotrypsin and subtilisin as in Dalle-Donne et al. [7]. Theextent of 3-NT formation was assessed either spectrophotometrically or byWestern-blotting as in Tiago et al. [5,6].Results and DiscussionIn vitro exposure of actin to micromolar peroxynitrite fluxes promoted an im-balance of the G-actin (monomer)/F-actin (filament) equilibrium which can effi-ciently block the stimulation of the myosin ATPase activity by actin. Furthermore,incubation of purified myosin with SIN-1 also resulted in inhibition of the myosinATPase activity stimulated by actin. The IC50 values, obtained from the nonlinearleast square fit of the data shown in Figure 1, take place at molar ratios of 6.12±0.86and 7.351.53 mol SIN-1/mol of myosin and actin, respectively, showing that bothproteins are highly sensitive to PN oxidation. The loss of myofibril actomyosinATPase activity parallels a decrease of the thermal stability of both proteins. Thishas been shown by the decrease of the unfolding enthalpy (DH) and critical tem-perature (Tc) of SIN-1 treated myosin and G-actin in the DSC experiments (Ta-ble 1). Moreover, the lack of significant decrease in the cooperative unit (DHV)189Vilamoura, Algarve, Portugal, October 10-13, 2007Figure 1. Inhibition of the myosin ATPase activity stimulated by actin upon SIN-1 treatment.Activity assays were done with F-actin (squares) or myosin subfragment-1 (circles) treatedfor 2h with different SIN-1 concentrations in 10mM Tris (pH 8.5), 25 mM KCl as indicatedin Materials and Methods.Figure 2. Molecular model of G-actin (PDB Id: 1J6Z) on the left, and myosin subfragment-1 (PDB Id: 2MYS) on the right, depicting the putative residues modified by PN. ATP andCa2+ represented as yellow and orange spheres, respectively, are shown just to indicate theactive site on the actin structure. Cyst and Met residues are represented in red and bluestick-and-balls, respectively. The 25kDa tryptic fragment of myosin is coloured green whereit is indicated in purple the putative nitrated Tyr residues.190 European Meeting of the Society for Free Radical Researchpoints out that PN is inducing partial unfolded states of myosin and G-actin whichcan be fully prevented by incubating the sample with GSH during SIN-1 treatment.To address the oxidative modifications accounting for the structural and functionalimpairment of the actomyosin complex under the oxidative stress induced by PN,different protein modifications commonly produced by PN were assessed as de-scribed in Materials and Methods. The results suggested a major role for Cys-707and Cys-697 on myosin, two highly reactive cysteines located close to the nucle-otide binding site (Figure 2) whose modification has been previously shown tohave dramatic effects on the functional properties of myosin. 3-NT formation couldbe detected in the 25 kDa tryptic myosin fragment (Figure 2), consistent with thelocation of the four nitrated tyrosines in the myosin heavy chain extracted fromcardiac muscle of aged rats [8]. However, the extent of this chemical modifica-tion is very low, i.e. less than 0.05 moles 3-NT/mole of myosin when 50% of theactivity is already lost. On actin, besides the highly reactive Cys-374, we found asignificant contribution of Met-44 and Met-47 to the observed impairment. WhileCys-374 is located at the C-terminal region which is the binding site for severalactin-binding proteins (including myosin), Met-44 and Met-47 are located in a loopimplicated in monomer/monomer contact (Figure 2). Therefore, the modificationof these critical residues by PN can directly affect the functional interaction withmyosin as well as the G-actin/F-actin equilibrium. 3-NT formation do not seemto significantly contribute to the actomyosin ATPase activity impairment.AcknowledgementsT. Tiago is supported by a post-doctoral grant (SFRH/BPD/20777/2004)from the Portuguese Foundation for Science and Technology (FCT). Workfunded by Grant 3PR05A078 of Junta de Extremadura.References[1] J. KANSKI et al. Proteomic analysis of protein nitration in aging skeletal muscleand identification of nitrotyrosine-containing sequences in vivo by nanoelectrosprayionization tandem mass spectrometry, J Biol Chem, 280, 24261-24266 (2005).[2] N.W. KOOY et al. Extensive tyrosine nitration in human myocardial inflamma-tion: evidence for the presence of peroxynitrite, Crit Care Med, 25, 812-819(1997).[3] M.J. MIHM et al. Impaired myofibrillar energetics and oxidative injury duringhuman atrial fibrillation, Circulation, 104, 174-180 (2001).[4] M.P. MURPHY et al. Peroxynitrite: a biologically significant oxidant, Gen Pharmacol,31, 179-186 (1998).[5] T. TIAGO et al. Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite,Biochemistry, 45, 3794-3804 (2006).[6] T. TIAGO et al. Peroxynitrite induces F-actin depolymerization and blockade ofmyosin ATPase stimulation, Biochem Biophys Res Commun 342, 44-49 (2006).[7] I. DALLE-DONNE et al. The tert-butyl hydroperoxide-induced oxidation of actinCys-374 is coupled with structural changes in distant regions of the protein, Bio-chemistry, 38, 12471-12480 (1999).[8] S.J. HONG et al. Proteomic analysis of age dependent nitration of rat cardiacproteins by solution isoelectric focusing coupled to nanoHPLC tandem massspectrometry, Exp Gerontol, 42, 639-651 (2007).

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