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Publications

Nitração de tirosinas na miosina: consequências funcionais

Publication . Palma, Pedro; Aureliano, M.

Tem sido demonstrado que o biomarcador 3-nitrotirosina (3-NT), frequentemente utilizado na reactividade do peroxinitrito (ONOO-) com as proteínas, aumenta na miosina após fibrilação atrial [Mihm et al, 2001b, 2003; Kooy et al, 1997] e durante o envelhecimento [Kansky et al, 2005a, 2005b; Hong et al, 2007].

2008Bachelor thesis

Open Access

Effects of reactive oxygen and nitrogen species on actomyosin and their implications for muscle contractility

Publication . Tiago, Teresa; Aureliano, M.; Gutiérrez-Merino, Carlos

Experimental evidence accumulated during recent years is pointing out that numerous pathological conditions in skeletal and cardiac muscle are associated with an oxidative stress-induced muscle injury. Additionally, it has been postulated that several oxidants can directly alter contractile function by oxidative modification of the myofibril proteins – actin and myosin. Peroxynitrite (ONOO-), a potent biological oxidizing agent formed in the nearly instantaneous reaction of nitric oxide with superoxide anion, is increasingly recognized as playing a major role in the skeletal and cardiac muscle dysfunction. This is supported by detection of 3-nitrotyrosine, a protein modification produced by the reaction of peroxynitrite with tyrosine, on skeletal and cardiac muscle proteins during aging or in diseases associated with myocardial inflammation or ischemia/reperfusion insults. Although some studies point to a correlation of protein nitration with functional and structural modifications, the mechanism by which peroxynitrite may impair muscle contractility remains far from being elucidated. In the present review we address the role of reactive oxygen and nitrogen species on the structural and functional impairment of actomyosin ATPase activity and their implications for muscle contraction with particular emphasis on the oxidative modifications promoted by peroxynitrite on actin and myosin.

2008Book part

Open Access

Actomyosin modulation by peroxynitrite

Publication . Tiago, Teresa; Silva, D.; Santos, Ana; Aureliano, M.; Gutiérrez-Merino, Carlos

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.

2007Conference object

Open Access

Efeito de peroxinitrito nos resíduos de actina

Publication . Silva, Dorinda; Aureliano, M.

Sabe-se que várias patologias estão associadas à existência de dano oxidativo nos tecidos. O peroxinitrito (ONOO-) é um dos agentes oxidantes referido em várias patologias, tais como na disfunção ventricular, na carcinogénese e em doenças neurodegenerativas. Para além destes estudos, verificou-se recentemente que a exposição dos filamentos de actina (F-actina) a fluxos submicramolares de peroxinitrito, provoca uma despolimerização destes filamentos com uma consequentemente inibição da estimulação da actividade ATPásica da miosina. No presente trabalho, estudou-se a susceptibilidade da actina, de músculo esquelético de coelho, à exposição crónica in vitro a peroxinitrito, resultante da decomposição do composto SIN-1. O SIN-1 liberta pulsos lentos das espécies oxidantes de superóxido (●O2-) e óxido nítrico (●NO), que reagem entre si resultando em peroxinitrito (ONOO-). O efeito deste agente oxidante na actina foi analisado medindo a capacidade de polimerização da proteína, a nitração dos resíduos de tirosina e a oxidação dos resíduos de cisteína e de metionina. Após exposição ao SIN-1 observou-se, que o peroxinitrito aumenta o tempo de meia vida (t1/2) da polimerização máxima de G-actina com um IC50 de 58,5 ± 10,4 µM SIN-1. Verificou-se oxidação de císteinas de G-actina, sendo a Cys374 a mais susceptível ao ONOO- (IC50 = 10,9 ± 2,5 µM SIN-1). Identificou-se ainda uma possível oxidação das metioninas nas posições 44 e 47 com valores de IC50 de 58,7 ± 19,2 μM e 30,8 ± 8,8 μM de SIN-1, respectivamente. Não se detectou, no entanto, formação de 3-nitrotirosinas. Apesar da oxidação da Cys374 preceder a oxidação da Met44 e da Met47, os valores de IC50 semelhantes encontrados para a inibição da polimerização da G-actina e para oxidação das metioninas, sugerem que estes resíduos apresentam um papel relevante na oxidação induzida pelo peroxinitrito na actina.

2006Bachelor thesis

Open Access

Monomeric versus decameric vanadate in the elucidation of muscle contraction regulation: a kinetic, spectroscopic and structural overview

Publication . Tiago, Teresa; Gutiérrez-Merino, Carlos; Aureliano, M.

Vanadium (V) was rediscovered for biology as a “muscle inhibitor factor” when it was found in commercial ATP prepared from equine muscle almost thirty years ago. Since then it has been used as a molecular probe of the mechanisms of several enzyme reactions involving hydrolysis of phosphate ester bonds. Besides acting as a phosphate analogue, vanadate has also the potential to exhibit biological activities through oligomeric vanadate species. Among the vanadate oligomers, decavanadate is one of the most potent inhibitors and has revealed an excellent kinetic and spectroscopic probe. This is particularly relevant for myosin, the major muscle ATPase which along with actin is able to convert the chemical energy of ATP hydrolysis into mechanical work. Apparently, vanadate is able to populate different conformational states of the myosin ATPase cycle depending on its oligomerization state. While monomeric vanadate (VO4 3-) mimics the transition state for the g-phosphate hydrolysis blocking myosin in a pre-power stroke state, decameric vanadate (V10O28 6-) induces the formation of the intermediate myosin·MgATP·V10 complex blocking the actomyosin cycle in a pre-hydrolysis state. These recent findings, that are now reviewed, point out to the importance of taking into account vanadate species variety in studies describing the interaction of vanadate with biological systems and incite the use of decavanadate as a biochemical tool to the elucidation of muscle contraction regulation.

2007Book part

Open Access