Young-Mi Go

Redox compartmentalization in eukaryotic cells

Diverse functions of eukaryotic cells are optimized by organization of compatible chemistries into distinct compartments defined by the structures of lipid-containing membranes, multiprotein complexes and oligomeric structures of saccharides and nucleic acids. This structural and chemical organization is coordinated, in part, through cysteine residues of proteins which undergo reversible oxidation-reduction and serve as chemical/structural transducing elements. The central thiol/disulfide redox couples, thioredoxin-1, thioredoxin-2, GSH/GSSG and cysteine/cystine (Cys/CySS), are not in equilibrium with each other and are maintained at distinct, non-equilibrium potentials in mitochondria, nuclei, the secretory pathway and the extracellular space. Mitochondria contain the most reducing compartment, have the highest rates of electron transfer and are highly sensitive to oxidation. Nuclei also have more reduced redox potentials but are relatively resistant to oxidation. The secretory pathway contains oxidative systems which introduce disulfides into proteins for export. The cytoplasm contains few metabolic oxidases and this maintains an environment for redox signaling dependent upon NADPH oxidases and NO synthases. Extracellular compartments are maintained at stable oxidizing potentials. Controlled changes in cytoplasmic GSH/GSSG redox potential are associated with functional state, varying with proliferation, differentiation and apoptosis. Variation in extracellular Cys/CySS redox potential is also associated with proliferation, cell adhesion and apoptosis. Thus, cellular redox biology is inseparable from redox compartmentalization. Further elucidation of the redox control networks within compartments will improve the mechanistic understanding of cell functions and their disruption in disease.

Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control

Redox mechanisms function in control of gene expression, cell proliferation, and apoptosis, but the circuitry for redox signaling remains unclear. Cysteine and methionine are the only amino acids in proteins that undergo reversible oxidation/reduction under biologic conditions and, as such, provide a means for control of protein activity, protein‐protein interaction, protein trafficking, and protein‐DNA interaction. Hydrogen peroxide and other reactive oxygen species (ROS) provide a mechanism to oxidize signaling proteins. However, oxidation of sulfur‐ containing side chains of cysteine and methionine by ROS can result in oxidation states of sulfur (e.g., sulfinate, sulfonate, sulfone) that are not reducible under biologic conditions. Thus, mechanisms for oxidation that protect against over‐oxidation of these susceptible residues and prevent irreversible loss of activity would be advantageous. The present study shows that the steady‐state redox potential of the cysteine/cystine couple (Eh = –145 mV) in cells is sufficiently oxidized (>90 mV) relative to the GSH/GSSG (–250 mV) and thioredoxin (Trx1, –280 mV) redox couples for the cysteine/cystine couple to function as an oxidant in redox switching. Consequently, the cysteine/cystine couple provides a means to oxidize proteins without direct involvement of more potent oxidants. A circuitry model incorporating cysteine as a redox node, along with Trx1 and GSH, reveals how selective interactions between the different thiol/disulfide couples and reactive protein thiols could differentially regulate metabolic functions. Moreover, inclusion of cysteine/cystine as a signaling node distinct from GSH and Trx1 significantly expands the redox range over which protein thiol/disulfide couples may operate to control physiologically relevant processes.

Cysteine/cystine redox signaling in cardiovascular disease

Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals. The major thiol/disulfide redox couple in human plasma is cysteine (Cys) and its disulfide form, cystine (CySS). Oxidation of this redox couple, measured as a more positive steady-state redox potential (E(h)), is associated with risk factors for cardiovascular disease (CVD), including aging, smoking, obesity, and alcohol abuse. Rodent and vascular cell studies show that the extracellular redox state of Cys/CySS (E(h)CySS) can play a vital role in controlling CVD through proinflammatory signaling. This inflammatory signaling is regulated by cell-surface protein redox state and involves mitochondrial oxidation, nuclear factor-κB activation, and elevated expression of genes for monocyte recruitment to endothelial cells. Gene array and proteomics studies reveal the global nature of redox effects, and different cell types, e.g., endothelial cells, monocytes, fibroblasts, and epithelial cells, show cell-specific redox responses with different phenotypic traits, e.g., proliferation and apoptosis, which can contribute to CVD. The critical nature of the proinflammatory redox signaling and cell biology associated with E(h)CySS supports the use of plasma levels of Cys, CySS, and E(h)CySS as key indicators of vascular health. Plasma redox-state-based pharmacologic interventions to control or improve E(h)CySS may be effective in preventing CVD onset or progression.

Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase

The c-Jun NH2-terminal kinase (JNK), also known as stress-activated protein kinase, is a mitogen-activated protein kinase that determines cell survival in response to environmental stress. Activation of JNK involves redox-sensitive mechanisms and physiological stimuli such as shear stress, the dragging force generated by blood flow over the endothelium. Laminar shear stress has antiatherogenic properties and controls structure and function of endothelial cells by mechanisms including production of nitric oxide (NO) and superoxide ([Formula: see text]). Here we show that both NO and [Formula: see text] are required for activation of JNK by shear stress in endothelial cells. The present study also demonstrates that exposure of endothelial cells to shear stress increases tyrosine nitration, a marker of reactive nitrogen species formation. Furthermore, inhibitors or scavengers of NO, [Formula: see text], or reactive nitrogen species prevented shear-dependent increase in tyrosine nitration and activation of JNK. Peroxynitrite alone, added to cells as a bolus or generated over 60 min by 3-morpholinosydnonimine, also activates JNK. These results suggest that reactive nitrogen species, in this case most likely peroxynitrite, act as signaling molecules in the mechanoactivation of JNK.

Redox Control Systems in the Nucleus: Mechanisms and Functions

Proteins with oxidizable thiols are essential to many functions of cell nuclei, including transcription, chromatin stability, nuclear protein import and export, and DNA replication and repair. Control of the nuclear thiol-disulfide redox states involves both the elimination of oxidants to prevent oxidation and the reduction of oxidized thiols to restore function. These processes depend on the common thiol reductants, glutathione (GSH) and thioredoxin-1 (Trx1). Recent evidence shows that these systems are controlled independent of the cytoplasmic counterparts. In addition, the GSH and Trx1 couples are not in redox equilibrium, indicating that these reductants have nonredundant functions in their support of proteins involved in transcriptional regulation, nuclear protein trafficking, and DNA repair. Specific isoforms of glutathione peroxidases, glutathione S-transferases, and peroxiredoxins are enriched in nuclei, further supporting the interpretation that functions of the thiol-dependent systems in nuclei are at least quantitatively distinct, and probably also qualitatively distinct, from similar processes in the cytoplasm. Elucidation of the distinct nuclear functions and regulation of the thiol redox pathways in nuclei can be expected to improve understanding of nuclear processes and also to provide the basis for novel approaches to treat aging and disease processes associated with oxidative stress in the nuclei.

Intracellular Proatherogenic Events and Cell Adhesion Modulated by Extracellular Thiol/Disulfide Redox State

Background—Oxidative stress, a contributing factor to atherosclerosis, causes oxidation of biological thiols, which can be quantified in terms of the thiol/disulfide redox. The major thiol/disulfide redox couple in human plasma is cysteine (Cys) and its disulfide, cystine (CySS). Although atherosclerosis has previously been associated with Cys/CySS oxidation, whether oxidation of Cys/CySS contributes in a causal way to atherosclerosis development is not known. We examined the function of extracellular Cys/CySS redox potential (Eh) in the regulation of early events of atherosclerosis using cultured aortic endothelial cells and monocytes as a vascular model system. Methods and Results—To determine the range of thiol/disulfide redox state in human plasma, we analyzed levels of Cys, CySS, glutathione (GSH), and glutathione disulfide (GSSG) and calculated Eh according to the Nernst equation. Eh of Cys/CySS and GSH/GSSG was −120 to −20 and −200 to −50 mV, respectively. To approximate this range, endothelial cells were exposed to initial Eh from −150 mV (most reduced) to 0 mV (most oxidized). Compared with more reduced Eh, oxidized Eh of Cys/CySS stimulated H2O2 but not nitric oxide production, activated nuclear factor-&kgr;B, increased expression of adhesion molecules (intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, P-selectin), and stimulated monocytes binding to endothelial cells. Extracellular Eh regulated thiol/disulfide redox states of extracellular membrane proteins and H2O2 production, indicating that variation in extracellular Eh is detected and signaled at the cell surface. Conclusions—The extracellular thiol/disulfide Eh of the Cys/CySS couple plays a key role in regulating early events of atherosclerosis and could be useful as a potential marker for vascular disease risk.

The Redox Proteome

The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges.

Mechanisms of Cell Signaling by Nitric Oxide and Peroxynitrite: From Mitochondria to MAP Kinases

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

After cellular internalization, quercetin causes Nrf2 nuclear translocation, increases glutathione levels, and prevents neuronal death against an oxidative insult.

In this work we describe the protective effects of quercetin against H(2)O(2) in 24-h-pretreated neuronal cultures. We explored quercetin availability and subcellular fate through the use of HPLC-Diode Array Detection (DAD), epifluorescence, and confocal microscopy. We focused on quercetin modulation of thiol-redox systems by evaluating changes in mitochondrial thioredoxin Trx2, the levels of total glutathione (GSH), and the expression of the gamma-glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH synthesis, by the use of Western blot, HPLC, and real-time PCR techniques, respectively. We further explored the activation of the protective NF-E2-related factor 2 (Nrf2)-dependent signaling pathway by quercetin using immunocytochemistry techniques. Our results showed rapid quercetin internalization into neurons, reaching the nucleus after its addition to the culture. Quercetin pretreatment increased total GSH levels, but did not increase Trx2. Interestingly it caused Nrf2 nuclear translocation and significantly increased GCLC gene expression. At the moment of H(2)O(2) addition, intracellular quercetin or related metabolites were undetectable in the cultures although quercetin pretreatment prevented neuronal death from the oxidant exposure. Our findings suggest alternative mechanisms of quercetin neuroprotection beyond its long-established ROS scavenging properties, involving Nrf2-dependent modulation of the GSH redox system.

Thiol/disulfide redox states in signaling and sensing

Abstract Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol–disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol–disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.