Redox-based regulation of signal transduction: principles, pitfalls, and promises

Abstract

Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.

Figure 1

Schematic overview of various reversible modifications of reactive cysteines (S⁻). S-S; disulfide, PSSG; glutathionylation, SNO; nitrosylation, SOH; sulfenic acid, SO₂H; sulfinic acid. Note that the arrows are strictly included for an illustrative purpose, and do not indicate directionality.

Figure 2

Schematic representation of mechanisms of redox regulation. Well described is the scenario where cysteine oxidation inactivates a signaling protein, such as a protein tyrosine phospatase (box 1) or caspases. Additionally, a protein with a signaling function can also be directly activated by oxidation of a critical cysteine residue (box 2). Examples of this scenario are Ras, ryanodine receptor, dynamin, SERCA, and Src tyrosine kinase ([50], for review). Another mechanism whereby cysteine oxidation elicits a signal is through control of multimerization (box 3), such as the dimerization of various heat shock or chaperone proteins or self-assembly of dynamin. Box 4 visualizes the situation wherein cysteine oxidation of a regulatory protein causes its dissociation of its partner, thereby activating the function of the partner. An example of this represents the dissociation of oxidized, thioredoxin from Ask1, leading to Ask1 activation, the dissociation from oxidized Keap-1 from NRF-2, leading the activation of NRF-2 as a transcription factor, or the dissociation of nNOS from glucokinase, leading to activation of the kinase [243]. Direct oxidation of transcription factors, may also be sufficient to mediate their activation or inhibiton through altered interactions with DNA (box 5). Examples include the activation of prokaryotic OxyR, which shifts from a “dimeric” to “tetrameric” interaction with DNA upon oxidation, and NF-κB (p50, p65) or cJun whose binding with DNA is inhibited by S-oxidation.

Figure 3

Reduction strategies used to detect various cysteine oxidations. These thiol trapping or biotin switch procedures involve sequential steps of thiol blocking (1), a wash step to remove the chemical blocking agents, followed by a reduction step (2). This reduction step can employ non-specific chemical reagents e.g. dithiotreitol (DTT) to decompose sulfenic acids, disulfides, S-nitrosylated cysteines, or S-glutathionylated cysteines (top). Alternatively, the reduction step utilizes ascorbate or UV to reduce S-nitroso bonds (middle). GRX-catalyzed reduction (bottom) is used to reduce S-glutathionylated proteins. In a labeling step (3), the newly reduced sulfhydryl groups are reacted with a thiol specific labeling agent. Subsequently, by capturing the labeled cysteines, proteins can be identified using proteomic procedures. Labeled proteins can be precipitated, electrophoresed and blotted, and specific antibodies can then be used to detect targets. These methods can also be used to evaluate reversible cysteine oxidations in situ, in cells or tissues, using microscopy approaches (See Figures 4 and 5). Alk denotes a thiol specific alkylating agent, such as N-ethyl maleamide (NEM).

Figure 4

In situ visualization of S-nitrosylated proteins in lung expithelial cells using confocal laser scanning cytometry. Cells were exposed to 1mM SNAP or GSNO for 1 hour, or to the NOS inhibitor, L-NMMA, or reagent control D-NMMA. As additional controls, the biotin-HPDP label (-label), or ascorbate (-Asc) were omitted, or HgCl₂ (which depletes SNO), was added before blocking to decompose S-nitrosothiols. As an additional control, cells were treated with H₂O₂ for 15 minutes (See [55] for experimental details). Reprinted from Nitric Oxide, Volume 11, In situ detection and visualization of S-nitrosylated proteins following chemical derivatization: Identification of Ran GTPase as a target for S-nitrosylation, Ckless K. et al., 216–227, Copyright 2004, with permission from Elsevier.

Figure 5

In situ visualization of S-glutathionylated proteins in lung expithelial cells using GRX-catalyzed cysteine derivatization, and visualization via confocal laser scanning cytometry. Top panels: Lung epithelial cells were left untreated and as reagent controls, GRX (-GRX) or MPB (-MBP) were omitted out of the procedure. As an additional control, cells were treated with 1mM DTT prior to blocking with NEM (preDTT). Bottom panels: During the reversal of cysteine oxidations by GRX, fully reduced Bovine Serum Albumin (BSA), Cysteinylated BSA (BSA-cys) or S-glutathionylated BSA (BSA-SSG) were co-incubated in the reaction mixture to evaluate competition with endogenous substrates for GRX1-catalyzed reduction. (See [100] for experimental details). Protein S-glutathionylation is visualized in green, and nuclei are indicated in red. Reprinted from Biochimica et Biophysica Acta, Volume 1760, In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization. Reynaert N. et al., 380–387, Copyright 2006, with permission from Elsevier.

Figure 6

Visualization of the differential impact of compartmentalized cysteine oxidation events, as compared to diffuse oxidation events on signal transduction cascades. The left panel represents a hypothetical schematic illustrating the compartmentalized cysteine oxidation events, S1-Ox, and S2-Ox, following the localized activation of NOX or NOS, which lead to activation of regulator X, which in turn evokes response X. The panel on the right represents diverse intracellular cysteine oxidation events S1-Ox, S3-Ox, and S4-Ox that occur in response to extracellularly encountered H₂O₂ or NO/SNO. This scenario leads to a different biological response (Z). Activation of Regulator X does not occur, because of lack of oxidation S2, and the oxidation of S3 which activates regulator Y, which in turn inhibits Pathway X. Instead, because of oxidation event S4, activation of regulator Z occurs, leading to activation of pathway Y, and response Z. The scenarios illustrated are strictly hypothetical.