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Amy Milsted Molecular Biology Spring 2013 Glutaredoxins: Structures, Biological Functions, and Significance Glutaredoxins are a group of redox enzymes which can bind and utilize the tripeptide glutathione as a substrate. Glutathione, a tripeptide of sequence GSH (Glycine-Serine-Histidine), is a major thiol compound which acts as a redox buffer and essential antioxidant. In response to free radicals and other compounds, glutathione may donate an electron to reduce the species and thus become oxidized. In its oxidized form, glutathione forms a homodimer called glutathione disulfide (GSSG). To regenerate glutathione, most organisms reduce GSSG through the dimeric enzyme, glutathione reductase, at the expense of NADPH (Lillig, 2008). In this glutathione system, glutaredoxin can become oxidized by a substrate and then reduced by glutathione. Although its role in mediating oxidative stress is the most commonly known feature of glutaredoxins, recent research has revealed that glutaredoxins are a versatile class of proteins with implications in DNA synthesis, protein folding, metal detoxification, electron transfer, and assembly of iron-sulfur clusters. Glutaredoxins are such a versatile class that they are expressed in virtually all organisms, tissues, cell types, and organelles (Hanschmann et al., 2013). The structure of glutaredoxins have been studied using both X-ray crystallography and NMR spectroscopy. Structurally, glutaredoxins belong to the thioredoxin fold family of proteins - bacterial glutaredoxins are most representative of this fold, which consists of a four-stranded beta sheet surrounded by three alpha helices (Lillig, 2008). Traditionally, glutaredoxins were classified into either one of two types: dithiol and monothiol glutaredoxins. Dithiol motifs contain a CPYC (Cysteine-ProlineTyrosine-Cysteine) active site whereas monothiol motifs contain a CGFS (Cysteine-GlycinePhenylalanine-Serine) active site, which lacks the C-terminal active site thiol but still retains the ability to bind and use glutathione as a substrate (Strher & Miller, 2012). Monothiol glutaredoxins can further be classified into single-domain monothiols, found in all kingdoms, and multi-domain monothiols, found only in eukaryotes (Lillig et al., 2008). The distinction between monothiol and dithiol motifs is sufficient to describe the limited number of glutaredoxins found in bacteria, yeast, and mammals; however, recent findings of many more glutaredoxins in higher order land plants have prompted new classifications according to sequence structure and functional types, with at least six new subclasses for glutaredoxins. Some proposed functional types include glutaredoxins for deglutathionylation, dimerization for cluster transport, and for transcriptional activation (Strher & Miller, 2012). Glutaredoxins were originally identified by their ability to deliver electrons to ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides and therefore provide the building blocks for DNA synthesis. Glutaredoxins may also serve as an electron donor in cases where thioredoxin is not present or abundant. A study has showed that E. coli mutants lacking thioredoxin activity can utilize glutaredoxin to reduce inorganic sulfate to sulfide, which are then used to synthesize compounds such as cysteine, methionine, and various sulfur-containing cofactors (Lillig et al., 2008). The ability of glutaredoxin to donate electrons also plays a role in arsenic resistance by serving as an electron donor to the ArsC protein family, which catalyzes the reduction of arsenate to arsenite (Lpez-Maury et al., 2009). Glutaredoxins play a prominent role in heavy metal detoxification through similar mechanisms. Plants, which contain significantly higher amounts of glutaredoxins than bacteria, mammals, and yeast, also utilize glutaredoxins as an electron donor in peroxides, methionine sulfoxides, and thioredoxins (Rouhier et al., 2008).

Oxidative stress can initiate redox-dependent modifications in thiol-containing proteins. Susceptible thiol groups are mainly found in cysteines, but the thiols of methionines and selenocysteines are also sometimes implicated. Thiols are susceptible to oxidative modifications, which can ultimately affect protein function. Small protein thiols or peptides such as glutathione can form disulfides with these modified proteins; a protein forming a disulfide bond with glutathione is a form of post-translational modification termed glutathionylation, which can result in either activation or inhibition of a variety of proteins. Glutaredoxins have been found to catalyze both the formation and reduction of such disulfides (Hanschmann et al., 2013). Glutathionylation and deglutathionylation reactions can occur spontaneously through various mechanisms, and most commonly occur when production of reactive oxygen species is enhanced. As of 2009, proteomic studies involving 35S-labeled glutathione have identified approximately 150 diverse proteins that can undergo glutathionylation, which are involved in processes such as glycolysis, signal transduction, protein degradation, intracellular trafficking, and protein folding (Rouhier et al., 2008). Glutaredoxins are responsible for deglutathionylation, which can alter protein activity, therefore implicating their importance in this wide variety of fundamental biological processes. Some dithiol and almost all of the monothiol glutaredoxins have been found to coordinate iron-sulfur clusters. To coordinate a [2Fe-2S] cluster, a glutaredoxin will form a homodimer bound to iron, with two glutathione molecules covalently linked to the cluster (Rouhier et al., 2010). The clusters, which are rather labile, can rapidly be transferred to acceptor proteins. Single domain monothiol glutaredoxins, located in mitochondria or chloroplasts, have been implicated in the maturation of iron-sulfur cluster proteins whereas multidomain monothiol glutaredoxins, located in the cytosol or nucleus, are thought to have roles in iron trafficking and regulation (Li & Outten, 2012). The involvement of glutaredoxins in iron-sulfur assembly implicate this protein group as scaffold proteins for de novo synthesis and transfer of the clusters, delivery proteins, and possibly regulators (Rouhier et al., 2008). Iron-sulfur clusters are essential to many processes, including photosynthesis, respiration, and assimilation of nitrogen and sulfur in plants. Mammalian glutaredoxins have been implicated in various pathways, such as apoptosis and cell proliferation. Glutaredoxin, along with thioredoxin, acts as a negative regulator of apoptosis signalregulating kinase 1 (ASK1). Binding of glutaredoxin and thioredoxin to ASK1 results in an inactive complex, which can be oxidized to activate ASK1 and result in a downstream signal to induce apoptosis (Hanschmann et al., 2013). Different types of glutaredoxins can have different effects on apoptosis. For example, glutaredoxin-1 activity is significantly upregulated by tumor necrosis factor-alpha in endothelial cells, which can result in glutathionylation and therefore activation of the caspase-3 protein, leading to apoptosis. On the other hand, glutaredoxin-2 has been shown to be capable of protecting HeLa cells from apoptosis induced by oxidative stress by attenuating release of cytochrome c and caspase activation (Lillig et al., 2008). These various roles that glutaredoxins play in fundamental biological processes implicate it in diseases throughout various tissues and organs. For example, embryonic development and cells of the nervous system are affected by oxygen concentrations and levels of reactive oxygen species, which are mediated by glutaredoxins. Iron-sulfur clusters and heme biosynthesis are also important in embryonic development, and low levels of glutathione in embryonic cells can lead to apoptosis over cell proliferation (Hanschmann et al., 2013). Sensory organs have been shown to express glutaredoxins. Eye pathologies, such as diabetic retinopathy and glaucoma, are associated with oxidative stress, inflammation, and glutathione levels which all implicate glutaredoxins. In the cardiovascular system, glutaredoxin-1 knockout mice showed increased infarct size and inhibition of functional recovery whereas overexpression of glutaredoxins resulted in reduced infarct size, protection against

cardiotoxicity, and inhibition of cardiac hypertrophy (Hanschmann et al., 2013). Glutaredoxins have also been found throughout skin, hair, and respiratory systems, as they are commonly exposed to chemical and physical modifications and pollutants. By studying the changes in glutaredoxin expression throughout various pathologies, researchers hope to utilize glutaredoxins as biomarkers to identify, diagnose, and determine the state of a disease. The role of glutaredoxins in infection and the immune system is of great importance to researchers. Certain viruses encode their own glutaredoxins, which are then used for DNA synthesis, formation of disulfide bonds, and viral assembly (Lillig et al., 2008). Viral glutaredoxins may also serve to protect the virus from oxidative stress as it enters the host cell. Human glutaredoxin has been found on the surface of HIV-1 and studies have indicated that it is important for HIV-1 entry (Hanschmann et al., 2013). Thus, current therapeutic targets are being researched to block the activity of these extracellular glutaredoxins, which would inhibit HIV-1 entry. Other forms of research involve exploiting the structural differences between bacterial and mammalian glutaredoxins to design and engineer potential drug delivery systems Although glutaredoxins were originally known for their role as redox enzymes, emerging studies demonstrate the importance of glutaredoxins in many biological functions and aspects. Glutathionylation implicates glutaredoxins in a wide variety of functions, such as energy metabolism, ion channel activity, redox signaling, transcription, cytoskeletal activity, and protein folding. Because deglutathionylation is unique to glutaredoxins, it is assumed that a majority of glutathionylated proteins involve glutaredoxins whose mechanisms are still yet to be discovered (Lillig & Berndt, 2012). The classification of glutaredoxins has proven to be more complicated than previously thought, and differs for different types of tissues and cells. Further studies of glutaredoxins will improve techniques in studying redox signaling in vivo, as the nature of the interactions are very transient and volatile, thus making them currently difficult to study (Hanschmann et al., 2013; Lillig & Berndt, 2012). In learning more about the structures and functions of glutaredoxins, glutaredoxins may also be designed and engineered for biological and clinical applications, regulatory mechanisms, or drug delivery systems. The emerging roles of glutaredoxins, which are continually being discovered, indicate that glutaredoxin has essential biological functions beyond that of a cellular redox buffer and there is much that continues to be learned about them. References Hanschmann, E., Godoy, J. R., Berndt, C., Hudemann, C., & Lillig, C. H. (2013). Thioredoxins, Glutaredoxins, and Peroxiredoxins - Molecular Mechanisms and Health Significance: From Cofactors to Antioxidants to Redox Signaling. Antioxidants and Redox Signaling. Ahead of print. doi:http://dx.doi.org/10.1089/ars.2012.4599 Li, H. & Outten, C. E. (2012). Monothiol CGFS Glutaredoxins and BoIA-like Proteins: [2Fe-2S] Binding Partners in Iron Homeostasis. Biochemistry, 51 (22): 4377-4389. doi:http://dx.doi.org/10.1021/bi300393z Lillig, C. H. & Berndt, C. (2012). Glutaredoxins in Thiol/Disulfide Exchange. Antioxidants and Redox Signaling. Ahead of print. doi:http://dx.doi.org/10.1089/ars.2012.5007 Lillig, C. H., Berndt, C., & Holmgren, A. (2008). Glutaredoxin systems. Biochimica et Biophysica Acta, 1780 (11), 1304-1314. doi:http://dx.doi.org/10.1016/j.bbagen.2008.06.003 Lpez-Maury, L., Snchez-Riego, A. M., Reyes, J. C., & Florencio, F. J. (2009). The Glutathione/Glutaredoxin System is Essential for Arsenate Reduction in Synechocystis sp. Strain PCC 6803. Journal of Bacteriology, 191 (11): 3534. doi:http://dx.doi.org/10.1128/JB.01798-08. Rouhier, N., Couturier, J., Johnson, M. K., & Jacquot, J. (2010). Glutaredoxins: roles in iron homeostasis. Trends in Biochemical Sciences, 35 (1): 43. doi:http://dx.doi.org/10.1016/j.tibs.2009.08.005

Rouhier, N., Lemaire, S. D., & Jacquot, J. (2008). The Role of Glutathione in Photosynthetic Organisms: Emerging Functions for Glutaredoxins and Glutathionylation. Annual Review of Plant Biology, 59: 143-166. doi:http://dx.doi.org/10.1146/annurev.arplant.59.032607.092811 Strher, E. & Millar, A. H. (2012). The biological roles of glutaredoxins. Biochemical Journal, 446, 333348. doi:http://dx.doi.org/10.1042/BJ20112131