Molecular mechanisms and clinical implications of reversible protein S-glutathionylation

Abstract

Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol-disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein-SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein-SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein-SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein-thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases.

FIG. 1.

Potential mechanisms of protein S-glutathionylation. This figure depicts various biochemical mechanisms by which protein thiol moieties could be converted to protein–SSG mixed disulide adducts. (1) via thiol-disulfide exchange; (2) via sulfenic acid intermediates; (3) via sulfenyamide intermediates; (4) via thiyl radical intermediates; (5) via thiosulfinate intermediates; (6) via S-nitrosyl intermediates. (See text for further explanation).

FIG. 2.

Glutaredoxin catalytic mechanism. This figure depicts glutaredoxin-catalyzed deglutathionylation of protein-SSG mixed disulfides. The central portion shows Grx catalysis proceeding via a monothiol mechanism involving a selective double displacement reaction. The glutathionylated sulfur moiety of the protein–SSG is attacked by the thiolate anion of the enzyme (Grx-S⁻), forming the covalent enzyme intermediate (GRx-SSG) and releasing the reduced protein-SH as the first product. The second rate-determining step involves reduction of the Grx-SSG by GSH to produce glutathione disulfide (GSSG) as the second product, recycling the reduced enzyme (Grx-S⁻). The left side depicts a dithiol catalytic mechanism which has also been proposed; however the monothiol mechanism is more prevalent and favored by a preponderance of evidence (see text). The right side depicts the side reaction involving formation of an intramolecular disulfide at the active site of the enzyme (C22-SS-C25) which detracts from catalysis (see text).

FIG. 3.

S-glutathionylation and GRx-regulation of proteins involved in insulin secretion (1a–6a) and insulin signaling (6b–12b). S-glutathionylation has been implicated in regulation of aldose reductase (1a), SERCA calcium channels (3a), PKC (4a), NF-κB (5a and 12b), PTP-1B (6b), Ras (7b), MEKK (8b), c-Jun (9b), Akt (10b), and IKK (11b). Grx has been reported to be involved in potassium channel gating (2a) and insulin secretion (6a).

FIG. 4.

Downstream effects of Ras glutathionylation in response to endogenous H₂O₂ production. Two independent modes of Ras activation by glutathionylation are depicted here. On the left is shown how Ras-dependent and -independent pathways contribute to angiotensin II-induced hypertrophy in vascular smooth muscle cells (Adachi et al., 2004), namely (1) coupling of angiotensin II receptor activation to production of ROS by NADPH oxidase, followed by Ras glutathionylation and activation of Akt and p38, and (2) ROS-independent transactivation of EGFR and activation of the ERK signaling pathway. The right-hand scheme depicts a mechanism by which Ras-SSG mediates the hypertrophic response of cardiomyocytes to mechanical strain. Strain-stimulated cardiac myocytes exhibit ROS-dependent Ras glutathionylation, which activates the ERK pathway and results in increased protein synthesis. The basis for activation of Akt and p38 (left) vs. ERK (right) pathways by the same signaling intermediate (i.e., Ras-SSG) is not yet understood.

FIG. 5.

Downstream effects of Ras glutathionylation in response to exogenous peroxinitrite or oxLDL. This figure depicts distinct downstream events resulting from Ras glutathionylation in bovine aortic endothelial cells in response to peroxinitrite added exogenously (left) or generated endogenously in response to oxLDL exposure (right) as reported by Clavreul et al. (47, 48). In response to ONOO⁻ treatment, Ras glutathionylation leads to transient phosphorylation of ERK and Akt; however, subsequent downstream signaling events remain unknown. oxLDL exposure (which leads to ONOO⁻ production in situ) also caused Ras-SSG-dependent, transient Akt phosphorylation (center); however, it induced a sustained time course of ERK phosphorylation (right) as well as diminished Akt activation in response to a subsequent stimulus (insulin). The decreased insulin-induced Akt activation conferred by oxLDL pretreatmentcould be explained by ERK-induced phosphorylation (and inactivation) of IRS, which is upstream of Akt in the insulin signaling pathway.

FIG. 6.

Evidence for potential roles of protein glutathionylation and Grx activity in lung diseases. This figure depicts the major conclusions of studies focused on protein glutathionylation and/or Grx in specific lung diseases, or in cell lines derived from different regions of the lung.

FIG. 7.

Apoptosis/survival signaling pathways that are regulated by reversible S-glutathionylation. This figure displays key signal transduction pathways in which regulation by reversible glutathionylation of signaling intermediates has been implicated. Target proteins that may play regulatory roles, and for which alteration in function due to glutathionylation has been reported in some context, are designated as protein-SSG [e.g., protein phosphatase 2A (PP2A)-SSG, phosphatase and tensin homologue deleted from chromosome 10 (PTEN)-SSG, protein kinase C (PKC)-SSG].

FIG. 8.

S-glutathionylation as a regulator of the JNK and MAPK signaling pathways in cancer. This figure depicts several sites in the JNK pathway and in other signaling pathways in cancer cells where reversible S-glutathionylation may serve as a potential regulatory mechanism (e.g., Ras-SSG, MAPK/ERK kinase kinase 1 (MEKK1)-SSG, c-Jun-SSG, glutathione S-transferase (GST_pi-SSG).

FIG. 9.

Potential targets of S-glutathionylation that are involved in cancer development and apoptosis. This block diagram lists various potential glutathionylation targets (central portion of the figure) and depicts how they are implicated in cancer cell progression or apoptosis (see text for further explanation).

FIG. 10.

FIG. 10. Potential glutathionylation targets implicated in neurodegenerative diseases. This figure illustrates the variety of cellular compartments influenced by protein glutathionylation. Evidence for changes in function associated with glutathionylation is discussed in the text for each of the target proteins depicted here.

FIG. 11.

Glutathionylation targets within the proteasome pathway. Glutathionylation occurs at multiple levels of the proteasome pathway resulting in inhibition of function. This oxidant-induced post translational modification leading to deactivation of the protein degradation process could contribute to protein aggregation that is prevalent in neurodegenerative diseases, including formation of Lewy bodies (PD) and neurofibrillary tangles (AD).