Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Eaton, Philip; Byers, Helen L.; Leeds, Nicola; Ward, Malcolm; Shattock, Michael J.

doi:10.1074/jbc.m111454200

Cited by 167 publications

(139 citation statements)

References 41 publications

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“…It is also possible the methods we have used may not trap all forms of protein sulfenic acids with equal efficiency. Many of the proteins identified clearly possess cysteines susceptible to oxidation, because we and others have already found them to be susceptible to other forms of cysteine targeted oxidation, such as S-thiolation (17,34,35) or Snitrosylation (36). Immunoblot analysis showed the extent to which sulfenic acid derivatives are formed varies considerably between proteins.…”

Section: Influence Of Concentration and Duration Of H2o2 Administratimentioning

confidence: 87%

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Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

Saurin

Neubert

Brennan

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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271

277

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A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.heart ͉ oxidative stress ͉ myocardium ͉ redox signaling

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Section: Influence Of Concentration and Duration Of H2o2 Administratimentioning

confidence: 87%

“…Isolated bufferperfused heart preparations were as before (17). Controls were aerobically perfused for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

Saurin

Neubert

Brennan

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

271

277

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“…A number of mitochondrial proteins have been identified as being glutathionylated, including subunits of complex I (12) and complex IV (13,14), aconitase (15,16), and pyruvate dehydrogenase (17); glutathionylation of these proteins is usually associated with a decrease of their activities. Because of the disruption in protein function that GSSG can cause, cells generally pump GSSG out of the cytoplasm into the extracellular matrix.…”

Section: Pr-smentioning

confidence: 99%

Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates

Garcia

Han

Sancheti

et al. 2010

Journal of Biological Chemistry

164

152

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Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be ؊171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F 1 complex, ␣-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be ؊291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H 2 O 2 challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.

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“…Stamler and Hausladen, 1998). S-thiolation is another mechanism that needs consideration (e.g., Eaton et al, 2002).…”

Section: Biological Ros/rns Sensors: Technical Issuesmentioning

confidence: 99%

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

2003

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In the past few years, nuclear DNA damage-sensing mechanisms activated by ionizing radiation have been identified, including ATM/ATR and the DNA-dependent protein kinase. Less is known about sensing mechanisms for cytoplasmic ionization events and how these events influence nuclear processes. Several studies have demonstrated the importance of cytoplasmic signaling pathways in cytoprotection and mutagenesis. For cytoplasmic signaling, radiation-stimulated reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential activators of these pathways. This review summarizes recent studies on the chemistry of radiation-induced ROS/ RNS generation and emphasizes interactions between ROS and RNS and the relative roles of cellular ROS/ RNS generators as amplifiers of the initial ionization events. Cellular mechanisms for regulating ROS/RNS levels are discussed. The mechanisms by which cells sense ROS/RNS are examined in terms of how ROS/RNS modify protein structure and function, for example, interactions with metal-thiol clusters, protein tyrosine nitration, protein cysteine oxidation, S-thiolation and Snitrosylation. We propose that radiation-induced ROS are the initiators and that nitric oxide (NO ) or derivatives are the effectors activating these signal transduction pathways. In responding to cellular ionization events, the cell converts an oxidative signal to a nitrosative one because ROS are too reactive and unspecific in their reactions for regulatory purposes and the cell is equipped to precisely modulate NO levels.

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Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Cited by 167 publications

References 41 publications

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

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