Detection and Mapping of Widespread Intermolecular Protein Disulfide Formation during Cardiac Oxidative Stress Using Proteomics with Diagonal Electrophoresis

Brennan, Jonathan P.; Wait, Robin; Begum, Shajna; Bell, James; Dünn, Michael J.; Eaton, Philip

doi:10.1074/jbc.m403827200

Cited by 180 publications

(140 citation statements)

References 40 publications

(37 reference statements)

Supporting

Mentioning

132

Contrasting

Order By: Relevance

“…Likewise, hemoglobin has been shown to be a cellular antioxidant in erythrocytes, where the highly reactive thiol groups of this extremely abundant protein have been shown to intercept oxidative species more effectively than glutathione (35). Because the cellular concentration of different proteins may vary quite dramatically this could reflect properties of a subset highly abundant proteins such as cytoskeletal proteins, which have been shown to be glutathionylated during diamide stress (36,37).…”

Section: Protein Thiols Constitute a Redox-active Pool That Is Similamentioning

confidence: 99%

Quantifying the global cellular thiol–disulfide status

Hansen

Roth²,

Winther³

2009

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

370

307

View full text Add to dashboard Cite

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the ''cellular redox buffer''; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.cysteine ͉ glutathione ͉ protein B ecause of the thiol group (SH), cysteine is the most chemically reactive natural amino acid found in cells. SH is mainly found in proteins (PSH) and in low-molecular-mass metabolites such as the highly abundant glutathione (GSH). Two SH groups can be oxidized to form a disulfide bond, and, in the cell, disulfides are mainly found in proteins (PSSP), in glutathione disulfide (GSSG), or as mixed disulfides between protein and glutathione (PSSG). In addition, SH groups can be reversibly oxidized by reactive oxygen species to nitrosothiols or sulfenic acids. Higher oxidation states, such as sulfinic and sulfonic acids, are generally considered to be irreversible.Disulfides are important for protein structure and folding, and formation of disulfide bonds can regulate protein function (1). Furthermore, glutathionylation, here used to denote the formation of PSSG, has been implicated as a mechanism for protection against irreversible protein oxidation during oxidative stress (2).The cytosolic glutathione pool is highly reducing (3) and consequently, disulfide bond formation is infrequent and typically only takes place transiently during catalysis of redox processes. In contrast, disulfide bond formation is favored in other compartments of the cell, such as the endoplasmic reticulum (ER) and the intermembrane space of mitochondria (4). The overall distribution of thiols and disulfides in different cellular pools defines the global thiol-disulfide redox status of the cell. Maintenance of thioldisulfide redox homeostasis in different subcellular compartments is important, and failure to do so can be detrimental to the structure, stability and activity of variou...

show abstract

Section: Protein Thiols Constitute a Redox-active Pool That Is Similamentioning

confidence: 99%

Quantifying the global cellular thiol–disulfide status

Hansen

Roth²,

Winther³

2009

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

370

307

View full text Add to dashboard Cite

show abstract

“…The formation of disulfide bridges has been investigated using off-diagonal electrophoresis [29], and this resulted in the evidence of complex interprotein bridges. Other studies have aimed at the identification of disulfide bridges brought upon oxidative stress between protein thiols and glutathione [30].…”

Section: 3studies On Mammalian Systemsmentioning

confidence: 99%

Oxidative stress response: a proteomic view

Rabilloud¹,

Chevallet²,

Luche³

et al. 2005

Expert Review of Proteomics

View full text Add to dashboard Cite

The oxidative stress response is characterized by various effects on a range of biological molecules. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classical biochemical methods, the recent onset of proteomics methods have allowed to study the oxidative stress response on a much wider scale. The input of proteomics in the study of oxidative stress response or in the evidence of an oxidative stress component in biological phenomena is thus reviewed in this paper. 3/2107/ 07/2008, 17:07:49

show abstract

“…it is non-stoichiometric) and consequently is unlikely to have functional significance. However, it is notable that both GAPDH [38,59,[70][71][72] and actin [38,52,56,70,71,73] have been shown in several studies to be redox modulated. In contrast, when low-abundance proteins such as signalling molecules are identified using proteomic methods, it might indicate a significant likelihood that they are important players in the cellular response to oxidants, as discussed below.…”

Section: Cardiac Proteins Susceptible To Oxidative Ptmmentioning

confidence: 99%

“…Proteins can form interprotein disulfide bonds after oxidant stress, including molecular chaperone proteins, growth factors and signal transduction proteins [37]. Brennan et al [38] have demonstrated widespread intermolecular protein disulfide formation during cardiac oxidative stress. Intraprotein disulfide-bond formation between proximal or vicinal thiols also occurs in many proteins [39].…”

Section: Protein Oxidation Involved In Redox Signallingmentioning

confidence: 99%

“…Diagonal electrophoresis can be used to identify proteins that form interprotein disulfide bonds [38,60], and specific proteins that form interprotein disulfide can be studied by non-reducing SDS-PAGE with immunoblotting. Antibody- based techniques can be also used to study specified oxidative modifications to proteins, including glutathiolation, homocysteinylation, carbonylation (e.g.…”

Section: Monitoring Oxidant Stress Using Cysteine Redoxmentioning

confidence: 99%

See 1 more Smart Citation

Redox signalling in cardiovascular disease

Charles

Eaton

2008

Proteomics Clinical Apps

Self Cite

View full text Add to dashboard Cite

Oxidative stress has almost universally and unequivocally been implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress in cells and cardiovascular biology was once considered only in terms of injury, disease and dysfunction. However, it is now appreciated that oxidants are also produced in healthy tissues, and they function as signalling molecules transmitting information throughout the cell. Conversely, when cells move to a more reduced state, as can occur when oxygen is limiting, this can also result in alterations in the function of biomolecules and subsequently cells. At the centre of this 'redox signalling' are oxidoreductive chemical reactions involving oxidants or reductants post translationally modifying proteins. These structural alterations allow changes in cellular redox state to be coupled to alterations in cell function. In this review, we consider aspects of redox signalling in the cardiovascular system, focusing on the molecular basis of redox sensing by proteins and the array of post-translational oxidative modifications that can occur. In addition, we discuss studies utilising proteomic methods to identify redox-sensitive cardiac proteins, as well as those using this technology more broadly to assess redox signalling in cardiovascular disease.

show abstract

Detection and Mapping of Widespread Intermolecular Protein Disulfide Formation during Cardiac Oxidative Stress Using Proteomics with Diagonal Electrophoresis

Cited by 180 publications

References 40 publications

Quantifying the global cellular thiol–disulfide status

Quantifying the global cellular thiol–disulfide status

Oxidative stress response: a proteomic view

Redox signalling in cardiovascular disease

Contact Info

Product

Resources

About