Protein-thiol oxidation and cell death: regulatory role of glutaredoxins

Abstract

Significance: Glutaredoxin (Grx) is the primary enzyme responsible for catalysis of deglutathionylation of protein-mixed disulfides with glutathione (GSH) (protein-SSG). This reversible post-translational modification alters the activity and function of many proteins important in regulation of critical cellular processes. Aberrant regulation of protein glutathionylation/deglutathionylation reactions due to changes in Grx activity can disrupt both apoptotic and survival signaling pathways.

Recent advances: Grx is known to regulate the activity of many proteins through reversible glutathionylation, such as Ras, Fas, ASK1, NFκB, and procaspase-3, all of which play important roles in control of apoptosis. Reactive oxygen species and/or reactive nitrogen species mediate oxidative modifications of critical Cys residues on these apoptotic mediators, facilitating protein-SSG formation and thereby altering protein function and apoptotic signaling.

Critical issues: Much of what is known about the regulation of apoptotic mediators by Grx and reversible glutathionylation has been gleaned from in vitro studies of discrete apoptotic pathways. To relate these results to events in vivo it is important to examine changes in protein-SSG status in situ under natural cellular conditions, maintaining relevant GSH:GSSG ratios and using appropriate inducers of apoptosis.

Future directions: Apoptosis is a highly complex, tightly regulated process involving many different checks and balances. The influence of Grx activity on the interconnectivity among these various pathways remains unknown. Knowledge of the effects of Grx is essential for developing novel therapeutic approaches for treating diseases involving dysregulated apoptosis, such as cancer, heart disease, diabetes, and neurodegenerative diseases, where alterations in redox homeostasis are hallmarks for pathogenesis.

FIG. 1.

Potential mechanisms of protein glutathionylation. There are multiple possible mechanisms for the formation of protein-SS-glutathione mixed disulfides. These mechanisms involve the activation of a free thiol, either of the protein or glutathione to a sulfenic acid, or formation of sulfenylamide, thiyl radical, thiosulfinates, or S-nitrosyl intermediates. These potential mechanisms are discussed in previous reviews (27,68).

FIG. 2.

Mechanism of protein deglutathionylation catalyzed by glutaredoxin (Grx). The first step of the Grx deglutathionylation reaction is nucleophilic attack of the enzyme's thiolate anion on the glutathionyl sulfur atom of the mixed disulfide of the glutathionylated protein. This results in the formation of a reduced protein and a glutathionylated Grx intermediate. The enzyme intermediate then can form either an intramolecular disulfide (side reaction) or be reduced by glutathione (GSH) (step 2), regenerating Grx and forming glutathione disulfide (GSSG).

FIG. 3.

Overview of apoptotic signaling through tumor necrosis factor superfamily receptor 6 (Fas) and tumor necrosis factor receptor (TNFR). When Fas ligand (FasL) (CD95L) binds to Fas it initiates the formation of the death-inducing signaling complex (DISC) involving the association of Fas-associated death domain (FADD) and pro-caspase-8 (pro-casp-8). In the absence of caspase-8-FADD-like interleukin-1β-converting enzyme inhibitory protein (c-FLIP), pro-caspase-8 is activated and initiates the caspase proteolytic cascade, leading to apoptosis. DISC formation can also lead to the activation of the MKK7 pathway and jun N-terminal kinase (JNK) phosphorylation, resulting in apoptosis. There are other cell-type-specific pathways that are affected by FasL. One of these pathways involves Bid, which can become truncated Bid (tBID) leading to the release of cytochrome C, and subsequently caspase activation. FasL can also stimulate the IκB kinase (IKK)/NFκB pathway resulting in the phosphorylation of IκB, leading to proteasome degradation and the translocation of NFκB to the nucleus. NFκB is responsible for initiating transcription of proteins, such as Grx, iNOS, and TNFα. The NFκB pathway can also be activated through the binding of TNFα to the TNFR, acting through NIK or TAK1. Proteins in these pathways for which there is experimental evidence implicating that they are regulated by Grx and protein glutathionylation are indicated by an asterisk (*).

FIG. 4.

Signaling pathways mediated by rat sarcoma (Ras) and protein kinase B (Akt). Ras and Akt are key apoptosis mediators responsible for several regulatory pathways. There is experimental evidence to implicate that many of the proteins in these pathways as being regulated by Grx and protein glutathionylation, as indicated by an asterisk (*). Depending on the cellular insult, cell type, and protein target, alterations in protein glutathionylation may result in cell survival or apoptosis. Ras is responsible for mediating the Akt, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK)/extracellular signal-regulated kinase (ERK), and MKK/JNK pathways. Glutathionylated Ras is an active form of this protein that can lead to increased Akt activity and therefore cell survival via the nuclear factor kappa-light chain enhancer of activated B cells (NFκB) cascade. When Grx deglutathionylates Ras, this can result in activation of the MEKK pathway and inhibition of Akt, resulting in apoptosis. An upstream regulator of Akt is phosphatase and tensin homolog (PTEN), which is responsible for dephosphorylating PIP3, preventing the activation of Akt. PTEN-SSG is the inactive form of this protein, and is no longer able to dephosphorylate PIP3. Therefore, in the absence of Grx, PIP3 is phosphorylated and capable of activating Akt, resulting in cell survival. Like PTEN-SSG, protein kinase C (PKC) is also inhibited when it is glutathionylated. However, this protein modification prevents PKC's ability to phosphorylate and activate Akt, causing apoptosis. The downstream effectors of Akt include apoptosis signaling kinase (ASK1), Apaf-1/caspase-9, and the NFκB pathway. Many of these proteins can also be regulated by Grx and protein glutathionylation. Grx is capable of binding to ASK1 resulting in the inhibition of this protein and ultimately cell survival. Grx regulates the NFκB pathway via deglutathionylation of IKK and NFκB. Grx is implicated in many of the cellular survival/death signaling mechanisms, evident by the number of proteins potentially regulated by Grx and glutathionylation in these pathways.

FIG. 5.

Role of glutathionylation in the Fas/FasL pathway. In C10 epithelial cells, pro-caspase-8 is activated when c-FLIP dissociates from Fas, resulting in the formation of DISC and the activation of pro-caspase-3 (pro-casp-3). Caspase-3 (Casp-3) is capable of degrading Grx, which can increase the number of glutathionylated Fas proteins. Fas-SSG is more readily recruited into lipid rafts, facilitating and propagating this apoptotic signal leading to enhanced Grx degradation and apoptosis, as described in (3). It is important to note that an alternative role of Grx in promoting apoptosis was reported (76); namely, in response to TNFα stimulation of endothelial cells, Grx is activated and catalyzes deglutathionylation of pro-caspase-3, thereby enabling proteolytic cleavage and release of the active caspase 3. Pro-caspase-3-SSG, which may be formed from pro-caspase-3-SNO, is unable to be cleaved, preventing Grx degradation and Fas deglutathionylation, thereby preventing apoptosis.

FIG. 6.

Potential regulation by Grx1 and Grx2 of mitochondrial proteins implicated in apoptotic initiation. The separate localization of the mammalian Grx isozymes is shown, along with specific glutathionylation-prone regulatory targets [outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM)]. With oxidative stress associated with various diseases, the mitochondrial contents of Grx1 and Grx2 may be altered; however, the impact of such changes on specific oxidation-sensitive mitochondrial proteins and corresponding commitment to apoptosis represents an important avenue of investigation.

FIG. 7.

Various cellular signals [reactive oxygen species (ROS), cytokines, growth factors, etc.] can disrupt protein glutathionylation and deglutathionylation by Grx. The disruption in protein glutathionylation status may be caused by a number of different cellular insults, such as ROS, reactive nitrogen species (RNS), cytokines, or growth factors. Depending on the insult and the cell type, protein target, and cellular conditions, this can result in changes to the glutathionylation status of a protein from the normal, steady-state form. This disruption results in protein deglutathionylation by Grx or aberrant formation of protein-SSG—either of which can lead to dysregulated apoptosis/survival signaling, and contribute to diseases, such as cancer and diabetes.