Conceptualización de la personalidad Grit

1.6.1 Metabolism

Two key enzymes participating in energy metabolism pathways that are modified by glutathionylation are Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and - ketoglutarate dehydrogenase complex (KGDHC) (reviewed in [27, 134]. GAPDH, a glycolytic enzyme was shown to be inactivated by glutathionylation in an in vitro

system. Incubating the protein with hydrogen peroxide further enhanced the extent of inactivation. Mitochondrial KGDHC in rat liver was found to be inactivated by glutathionylation on exposure to hydrogen peroxide. It was also shown that the glutathionylation-dependent inhibition was reversed by glutaredoxin [135]. Other metabolic enzymes such as aldolase, triose phosphate isomerase and alcohol dehydrogenase have also been reported to be regulated by glutathionylation in yeast

Saccharomyces cerevisiae in response to oxidative stress [136].

1.6.2 Apoptosis

Post translational modification of proteins including transcription factors, histones and intermediary signalling molecules has been shown to interfere with the cell proliferation by the activation of apoptotic pathways. A classic example is the disruption of the cell cycle via the glutathionylation of PTP1B and Mitogen-activated protein kinase kinase kinase 1 (MAP3K1/MEKK1) in response to oxidative stress. PTP1B and MEKK1 have been shown to undergo glutathionylation at specific cysteines, which inactivates the enzymes causing adverse downstream effects [27, 29]. The thiol modification interferes with ATP binding which in turn affects the ability of the kinases to phosphorylate their target substrates.

20 Investigations of the glutathionylation of transcription factors and cell signalling molecules such as NF-κB, TNF-, p53, GTPase, p21 Ras and Caspase-3 by glutathionylation have revealed inhibitory effects on the cell cycle and on the activation of apoptotic signalling in response to oxidative stress and cytotoxic agents [27, 137].

1.6.3 Regulation of calcium ion channels

The two main calcium channels in muscles are prime targets for glutathionylation under oxidative stress. Sarcoplasmic reticulum calcium ATPase (SERCA) and Ryanodine receptors (RyR) located in the muscle sarcoplasmic reticulum are highly sensitive to the reducing environment generated in the SR even under normal physiological conditions [27]. Glutathionylation of these calcium channels adversely affects their activity during stressed conditions such as myocardial ischemia resulting in irregular intracellular uptake and release of calcium ions and an overall increase in cellular calcium levels [138-140]. The reversibility of glutathionylation dependent inhibition of function was also demonstrated by these studies

1.6.4 Redox regulation of epigenetic DNA modifications

A recently published study identified glutathionylation as an indirect mechanism of DNA manipulation via histone modification [141]. The study demonstrated the significance of glutathionylation in drug response by establishing a link between histone glutathionylation and reversal of resistance to Doxorubicin (Dx) in MCF7/Dx, a Doxorubicin-resistant breast cancer cell line. This is the first study to identify glutathionylation as a mechanism of histone modification that indirectly introduces a novel theory: Is GSTO1-1 translocated into the nucleus to modify nuclear proteins?

1.6.5 Cytoskeletal remodelling

Actin is found in abundance in almost all cell types and is heavily glutathionylated under normal conditions [27, 69]. The glutathionylation of actin plays a significant role in cytoskeletal organization that occurs during cell division and cell adhesion. Remodelling involves the polymerization of actin into filaments and the disassembly of actin-myosin complexes during adhesion, both of which are modulated by glutathionylation (reviewed in [27, 134]. The thiol modification of specific cysteine residues was also found to regulate the contractile activity of actin in the cardiac muscle during ischemic attacks. Paradoxically, oxidative stress induced by artificial agents in vitro was found to deglutathionylate actin in A-431 cancer cells affecting actin polymerization during cell division [69].

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1.6.6 Inflammation

Many diseases are modified by inflammation and glutathionylation appears to modify the function of many proteins involved in inflammatory pathways. The functional role that glutathionylation plays in several diseases exacerbated by inflammation such as diabetes, Alzheimer’s disease, cardiovascular diseases, atherosclerosis and cancer is well elucidated [27, 53, 75, 142-146]. The anti-microbial activity of reactive species generated excessively in response to the presence of pathogens is well-documented [147-150]. The cytosolic source of ROS in phagocytic cells such as macrophages has been attributed to the activity of the family of NADPH oxidases [149, 150]. The functional implications of ROS mediated glutathionylation of proteins involved in inflammatory responses may be the missing link between oxidative stress and inflammation. A few examples of the modulation of inflammation by glutathionylation of specific proteins are presented below:

a. Regulation of chemotaxis: The myeloid related proteins S100A8 and S100A9 are calcium-binding proteins that belong to the S100 family of pro-inflammatory proteins [151, 152]. They are abundantly expressed in phagocytic cells such as neutrophils and monocytes. Both proteins are considered as markers of acute inflammation and are secreted extracellularly during pro-inflammatory responses [153]. The pro- inflammatory activity of neutrophils stimulated by S100A8/A9 is initiated by the association of S100A8 and S100A9 and subsequent polymerization with actin in response to increased calcium levels [153]. This in turn enhances the adhesion and chemotaxis ability of neutrophils. The oxidative burst occurring in activated neutrophils is sufficient to induce the glutathionylation and inhibition of S100A9 [154]. The complex calprotectin, formed by the association of S100A8/A9, is required for the adhesion of neutrophils to fibronectin on activation. The increased ROS has been shown to induce the glutathionylation of S100A9 alone which in turn prevents the adhesion of neutrophils to endothelial cells by inhibiting the oxidation and oligomerization of S100A8/A9 in response to increased ROS. On the other hand, induction of glutaredoxin by inflammatory stimuli has been reported by several studies [21, 61, 62, 64, 75] which suggests that glutaredoxins are required to maintain the redox state of proteins under different inflammatory stimuli. More recently, Bowers et. al (2012) have demonstrated the inhibitory effect of Sulfiredoxin-mediated glutathionylation of S100A4 resulting in the disruption of the interaction of S100A4 with cytoskeletal component non-muscle

22 myosin (NMIIA) [155]. The inability of S100A4 to interact with NMIIA was shown to impact cytoskeletal remodelling and consequently cell adhesion and migration.

ROS produced in immune cells is also responsible for the increased glutathionylation measured by this study and in previously published studies. NOX4-mediated ROS caused an increase in the global levels of protein glutathionylation in monocytes treated with a combination of LDL and high concentrations of D-glucose [156]. The study identified actin as a target of ROS induced glutathionylation which in turn acts as a priming agent accelerating chemotaxis in metabolically stressed monocytes, suggesting a novel role for actin glutathionylation in metabolic disorders associated with increased atherogenesis [156, 157]. MAP kinase phosphatase-1 (MKP-1), another key protein involved in monocyte migration, is similarly glutathionylated in response to intracellular ROS [74]. Glutathionylation of both actin and MKP-1 enhance adhesion and migration of monocytes in response to metabolic stress. The ratio of F-actin to G- actin was also reduced by metabolic stress in unstimulated monocytes, an effect that was further enhanced by MCP-1 stimulation as a consequence of increased glutathionylation of actin [74, 157].

b. TNFα signalling in macrophages: Activation and differentiation of the murine monocytic M1 cell line by IL-6 has been shown to induce expression levels of glutaredoxin which correlated with increased phagocytosis in the cell line [158]. A recent study published by Salzano et al. elegantly demonstrated the glutathionylation and consequent secretion of peroxiredoxin by activated macrophages [159]. Glutathionylation was shown to be essential for the secretion of Prdx which in turn induced the secretion of TNFα in the macrophages. Prdx was co-released along with thioredoxin, raising the speculation that this system was in place to maintain the oxidation/reduction of cell surface receptors such as the Toll like receptors (TLRs). The recruitment and activation of several intermediates in the TLR signalling pathways in immune cells have been shown to be influenced by the redox state of the signalling proteins [61, 118, 129, 160]. TLRs mediate pro- and anti-inflammatory responses on activation via signalling cascades that require the recruitment and association of intermediate proteins, most of which are shared among the members of the TLR family (13 in mammals) [161-163].

c. Inhibition of NF-κB dependent transcription of inflammatory genes: Inflammatory responses mediated by TLRs result in the activation of the master regulator of

23 inflammatory genes, NF-κB by phosphorylation of its subunits p65 and p50 and subsequent nuclear translocation of the subunits in order to activate the transcription of inflammatory genes such as IL-6, TNFα, IL-1β, NADPH oxidase,etc [164-167]. The glutathionylation of NF-κB p50 subunit was shown to inhibit DNA binding by NF-κB [119]. The glutathionylation of upstream IKK complex subunit IKKβ has also been shown to modulate its ability to phosphorylate NF-κB in lung epithelial cells, which was reversible by glutaredoxin [118].

d. Modulation of TLR4 signalling: The downstream recruitment and activation of TNF receptor-associated factor 6 (TRAF6) is essential for LPS stimulated signalling via TLR4/IL-1R in macrophages and B-cells [168]. On LPS stimulation, TRAF6 undergoes K63-linked auto-polyubiquitination which is required for the downstream protein- protein interactions leading to the activation of NF-κB [61]. Under physiological conditions, TRAF6 remains glutathionylated and inactive. When stimulated with LPS, TRAF6 undergoes glutaredoxin-1 catalysed deglutathionylation and subsequent auto- polyubiquitination and activation. However, the identity of the cysteine(s) was not further investigated.

e. Doxorubicin induced protein glutathionylation: Treatment of human monocyte- derived macrophages with the anti-cancer drug doxorubicin resulted in an increase in global glutathionylation levels before causing cell death [169]. Knocking down glutaredoxin aggravated cell death in the macrophages, suggesting that the regulation of deglutathionylation by glutaredoxins is necessary to maintain the increased glutathionylation levels in response to doxorubicin [169]. Interestingly, macrophage dysfunction was reported in adriamycin treated mice though the glutathionylation profile in affected tissues was not investigated [170].

f. Regulation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex activity: The redox protein thioredoxin interacting protein (TXNIP) was shown to disassociate from thioredoxin and directly interact with NLRP3, activating the formation of the NLRP3 inflammasome complex though subsequent studies could not validate this finding further [171]. Caspase 1, a component of the NLRP3 inflammasome complex and a cysteine protease responsible for the proteolytic activation of IL-1β is inactivated by glutathionylation induced by excessive ROS on LPS stimulation [160]. Since NLRP3 activation plays a prominent role in obesity induced inflammation and insulin resistance [172], the modulation of Caspase-1

24 glutathionylation may provide a novel therapeutic avenue for potential anti- inflammatory drugs. The direct functional implications of protein glutathionylation on different proteins and inflammatory pathways has been extensively reviewed previously [142]. A more detailed discussion of previously published findings that are directly relevant to this study is presented in Chapter 9.