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SummaryPeroxiredoxins (Prdx) utilize reversibly oxidized cysteine residues to reduce peroxides but also to promote H2O2signal transduction, including H2O2-induced activation of P38 MAPK. Prdx form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here we show that a genetically-encoded fusion between Prdx and the P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. In yeast, we demonstrate that a P38-Prdx fusion protein compensates for the loss of a scaffold protein and upstream MAP3K kinase activity, driving entry into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides a scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx can be sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.HighlightsP38-Prdx complexes increase P38 (Sty1/MAPK14) phosphorylation in yeast and human cellsTheS. pombePrdx promotes transient thioredoxin-mediated oxidation of a MAPK tyrosine phosphataseP38-Prdx complexes increase P38(Sty1) activity by phosphatase and MAP3K-independent mechanismsP38-Prdx complexes increase the stability and phosphorylation of theS. pombeP38 MAPKK (Wis1)Non-canonical, H2O2-induced autophosphorylation contributes to activation of the Wis1 MAPKK

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment.

In order to protect against oxidative damage, cells have evolved a host of ROS-detoxifying enzymes. These include peroxiredoxins, a highly conserved family of thioredoxin peroxidases. Unexpectedly, given their role in lowering H2O2levels, peroxiredoxins have been shown to be required for the activation of conserved stress-activated MAPKs in response to ROS in yeast1 and human2 cells. For example, we have previously shown that the single 2-Cys peroxiredoxin in S. pombe, Tpx1, but not its thioredoxin peroxidase activity, is required for the H2O2-induced activation of the p38/JNK-related MAPK, Sty1. Our findings revealed that Tpx1 forms H2O2-induced disulphide bonds with cysteines in Sty11, which suggested that Tpx1 may directly regulate Sty1 through these complexes. However, the mechanisms by which Tpx1-Sty1 disulphide complexes alter Sty1 function have remained unclear. Sty1, like its mammalian counterparts, has a number of important functions, including roles in coordinating cell growth, division, stress resistance and longevity in response to a variety of nutritional and stress stimuli. Our data suggests that disulphide complexes with Tpx1 are important for a subset of these roles. Intriguingly, our proteomic studies have identified multiple protein kinases that form disulphide complexes with Tpx1, these include kinases with established roles in regulating cell division and ageing. Here, we will present data suggesting that interactions with Tpx1 play important roles in regulating the activities of these kinases. 1. Veal et al. (2004) Molecular Cell, 15(1), pp. 129-139. 2. Jarvis et al. (2012) Free Radical Biology and Medicine, 53(7), pp. 1522-1530.

Peroxiredoxins are highly conserved and abundant peroxidases. Although the thioredoxin peroxidase activity of peroxiredoxin (Prx) is important to maintain low levels of endogenous hydrogen peroxide, Prx have also been shown to promote hydrogen peroxide-mediated signalling. Mitogen activated protein kinase (MAPK) signalling pathways mediate cellular responses to a variety of stimuli, including reactive oxygen species (ROS). Here we review the evidence that Prx can act as both sensors and barriers to the activation of MAPK and discuss the underlying mechanisms involved, focusing in particular on the relationship with thioredoxin.

Reactive oxygen species (ROS), generated externally and during aerobic metabolism, are a potent cause of cell damage. Oxidative damage is a feature of many diseases and ageing, including age-associated diseases, such as diabetes, cancer, cardiovascular and neurodegenerative diseases. Indeed, this association helped lead to the widely expounded 'Free Radical Theory of Aging', proposing that the accumulation of ROS-induced damage is the underlying cause of ageing. In the last decade, it has become apparent that ROS play more complex roles in ageing than simply causing damage. This includes the induction of signalling pathways that protect against/repair cell damage. Cells encode a variety of enzymes that metabolise ROS, some of which reduce them to less reactive species. In this chapter, we review the evidence that manipulating the levels of these enzymes has any effect/s on ageing. We will also highlight a few examples illustrating why it is an over-simplification to describe the activities of some of these enzymes as 'antioxidants'. We discuss how these studies have helped refine our view of how ROS and ROS-metabolising enzymes contribute to the ageing process.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

In 2003, structural studies revealed that eukaryotic 2-Cys peroxiredoxins (Prx) have evolved to be sensitive to inactivation of their thioredoxin peroxidase activity by hyperoxidation (sulfinylation) of their peroxide-reacting catalytic cysteine. This was accompanied by the unexpected discovery, that the sulfinylation of this cysteine was reversible in vivo and the identification of a new enzyme, sulfiredoxin, that had apparently co-evolved specifically to reduce hyperoxidized 2-Cys Prx, restoring their peroxidase activity. Together, these findings have provided the impetus for multiple studies investigating the purpose of this reversible, Prx hyperoxidation. Recent Advances: It has been suggested that inhibition of the thioredoxin peroxidase activity by hyperoxidation can both promote and inhibit peroxide signal transduction, depending on the context. Prx hyperoxidation has also been proposed to protect cells against reactive oxygen species (ROS)-induced damage, by preserving reduced thioredoxin and/or by increasing non-peroxidase chaperone or signaling activities of Prx.Here, we will review the evidence in support of each of these proposed functions, in view of the in vivo contexts in which Prx hyperoxidation occurs, and the role of sulfiredoxin. Thus, we will attempt to explain the basis for seemingly contradictory roles for Prx hyperoxidation in redox signaling.We provide a rationale, based on modeling and experimental studies, for why Prx hyperoxidation should be considered a suitable, early biomarker for damaging levels of ROS. We discuss the implications that this has for the role of Prx in aging and the detection of hyperoxidized Prx as a conserved feature of circadian rhythms. Antioxid. Redox Signal. 28, 574-590.

Aims: As Candida albicans is the major fungal pathogen of humans, there is an urgent need to understand how this pathogen evades toxic reactive oxygen species (ROS) generated by the host immune system. A key regulator of antioxidant gene expression, and thus ROS resistance, in C. albicans is the AP-1-like transcription factor Cap1. Despite this, little is known regarding the intracellular signaling mechanisms that underlie the oxidation and activation of Cap1. Therefore, the aims of this study were; (i) to identify the regulatory proteins that govern Cap1 oxidation, and (ii) to investigate the importance of Cap1 oxidation in C. albicans pathogenesis. Results: In response to hydrogen peroxide (H2O2), but not glutathione-depleting/modifying oxidants, Cap1 oxidation, nuclear accumulation, phosphorylation, and Cap1-dependent gene expression, is mediated by a glutathione peroxidase-like enzyme, which we name Gpx3, and an orthologue of the Saccharomyces cerevisiae Yap1 binding protein, Ybp1. In addition, Ybp1 also functions to stabilise Cap1 and this novel function is conserved in S. cerevisiae. C. albicans cells lacking Cap1, Ybp1, or Gpx3, are unable to filament and thus, escape from murine macrophages after phagocytosis, and also display defective virulence in the Galleria mellonella infection model. Innovation: Ybp1 is required to promote the stability of fungal AP-1-like transcription factors, and Ybp1 and Gpx3 mediated Cap1-dependent oxidative stress responses are essential for the effective killing of macrophages by C. albicans. Conclusion: Activation of Cap1, specifically by H2O2, is a prerequisite for the subsequent filamentation and escape of this fungal pathogen from the macrophage. Antioxid. Redox Signal. 19, 2244–2260.

Eukaryotic 2-Cys peroxiredoxins (Prx) are abundant antioxidant enzymes whose thioredoxin peroxidase activity plays an important role in protecting against oxidative stress, aging, and cancer. Paradoxically, this thioredoxin peroxidase activity is highly sensitive to inactivation by peroxide-induced Prx hyperoxidation. However, any possible advantage in preventing Prx from removing peroxides under oxidative stress conditions has remained obscure. Here we demonstrate that, in cells treated with hydrogen peroxide, the Prx Tpx1 is a major substrate for thioredoxin in the fission yeast Schizosaccharomyces pombe and, as such, competitively inhibits thioredoxin-mediated reduction of other oxidized proteins. Consequently, we reveal that the hyperoxidation of Tpx1 is critical to allow thioredoxin to act on other substrates ensuring repair of oxidized proteins and cell survival following exposure to toxic levels of hydrogen peroxide. We conclude that the inactivation of the thioredoxin peroxidase activity of Prx is important to maintain thioredoxin activity and cell viability under oxidative stress conditions.

Summary Endogenous and exogenous stresses elicit transcriptional responses that limit damage and promote cell/organismal survival. Like its mammalian counterparts, hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator‐activated receptor α (PPARα), Caenorhabditis elegans NHR‐49 is a well‐established regulator of lipid metabolism. Here, we reveal that NHR‐49 is essential to activate a transcriptional response common to organic peroxide and fasting, which includes the pro‐longevity gene fmo‐2/flavin‐containing monooxygenase. These NHR‐49‐dependent, stress‐responsive genes are also upregulated in long‐lived glp‐1/notch receptor mutants, with two of them making critical contributions to the oxidative stress resistance of wild‐type and long‐lived glp‐1 mutants worms. Similar to its role in lipid metabolism, NHR‐49 requires the mediator subunit mdt‐15 to promote stress‐induced gene expression. However, NHR‐49 acts independently from the transcription factor hlh‐30/TFEB that also promotes fmo‐2 expression. We show that activation of the p38 MAPK, PMK‐1, which is important for adaptation to a variety of stresses, is also important for peroxide‐induced expression of a subset of NHR‐49‐dependent genes that includes fmo‐2. However, organic peroxide increases NHR‐49 protein levels, by a posttranscriptional mechanism that does not require PMK‐1 activation. Together, these findings establish a new role for the HNF4/PPARα‐related NHR‐49 as a stress‐activated regulator of cytoprotective gene expression.

MAPK are activated by and orchestrate responses to multiple, diverse stimuli. Although these responses involve the increased phosphorylation of substrate effector proteins, e.g. transcription factors, the mechanisms by which responses are tailored to particular stimuli are unclear. In the fission yeast Schizosaccharomyces pombe, the Sty1 MAPK is crucial for changes in gene expression that allow adaptation to many forms of environmental stress. Here, we have identified two cysteine residues in Sty1, Cys-153 and Cys-158, that are important for hydrogen peroxide-induced gene expression and oxidative stress resistance but not for other functions of Sty1. Many Sty1-dependent changes in gene expression are mediated by the Atf1 transcription factor. In response to stress, Sty1 increases Atf1 levels by (i) promoting increases in atf1 mRNA and by (ii) directly phosphorylating and stabilizing Atf1 protein. Although dispensable for phosphorylation and stabilization of Atf1 protein, we find that both Cys-153 and Cys-158 are required for increases in atf1 mRNA levels and Atf1-dependent gene expression in response to hydrogen peroxide but not osmotic stress. Indeed, our data indicate that oxidation of Sty1, by formation of a disulfide bond between Cys-153 and Cys-158, is important for maintaining atf1 mRNA stability at high concentrations of hydrogen peroxide. Together, these data reveal that redox regulation of cysteine thiols in Sty1 is involved in a stress-specific mechanism regulating transcriptional responses to oxidative stress. Intriguingly, the conservation of these cysteine residues in other MAPK raises the possibility that similar mechanisms may ensure appropriate responses to hydrogen peroxide in other eukaryotes.

Oxidative damage caused by reactive oxygen species (ROS) is implicated in many diseases and in aging. Removal of ROS by antioxidant enzymes plays an important part in limiting this damage. For instance, peroxiredoxins (Prx) are conserved, abundant, thioredoxin peroxidase enzymes that function as tumor suppressors. In addition to detoxifying peroxides, studies in single-cell systems have revealed that Prx act as chaperones and redox sensors. However, it is unknown in what manner the different activities of Prx influence stress resistance or longevity in the context of whole animals. Here, we reveal three distinct roles for the 2-Cys Prx, PRDX-2, in the stress resistance of the nematode worm Caenorhabditis elegans . ( i ) The thioredoxin peroxidase activity of PRDX-2 protects against hydrogen peroxide. ( ii ) Consistent with a chaperone activity for hyperoxidized PRDX-2, peroxide-induced oxidation of PRDX-2 increases resistance to heat stress. ( iii ) Unexpectedly, loss of PRDX-2 increases the resistance of C. elegans to some oxidative stress-causing agents, such as arsenite, apparently through a signaling mechanism that increases the levels of other antioxidants and phase II detoxification enzymes. Despite their increased resistance to some forms of oxidative stress, prdx-2 mutants are short-lived. Moreover, intestinal expression of PRDX-2 accounts for its role in detoxification of exogenous peroxide, but not its influence on either arsenite resistance or longevity, suggesting that PRDX-2 may promote longevity and protect against environmental stress through different mechanisms. Together the data reveal that in metazoans Prx act through multiple biochemical activities, and have tissue-specific functions in stress resistance and longevity.

It is well established that oxidative stress is an important cause of cell damage associated with the initiation and progression of many diseases. Consequently, all air-living organisms contain antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species, including hydrogen peroxide. However, in eukaryotes, hydrogen peroxide also has important roles as a signaling molecule in the regulation of a variety of biological processes. Here, we will discuss the molecular mechanisms by which hydrogen peroxide is sensed and the increasing evidence that antioxidant enzymes play multiple, key roles as sensors and regulators of signal transduction in response to hydrogen peroxide.

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.

As a more selectively reactive oxygen species, H2O2 (hydrogen peroxide) has been co-opted as a signalling molecule, but high levels can still lead to lethal amounts of cell damage. 2-Cys Prxs (peroxiredoxins) are ubiquitous thioredoxin peroxidases which utilize reversibly oxidized catalytic cysteine residues to reduce peroxides. As such, Prxs potentially make an important contribution to the repertoire of cell defences against oxidative damage. Although the abundance of eukaryotic 2-Cys Prxs suggests an important role in maintaining cell redox, the surprising sensitivity of their thioredoxin peroxidase activity to inactivation by H2O2 has raised questions as to their role as an oxidative stress defence. Indeed, work in model yeast has led the way in revealing that Prxs do much more than simply remove peroxides and have even uncovered circumstances where their thioredoxin peroxidase activity is detrimental. In the present paper, we focus on what we have learned from studies in the fission yeast Schizosaccharomyces pombe about the different roles of 2-Cys Prxs in responses to H2O2 and discuss the general implications of these findings for other systems.

The adenovirus E1A protein both activates and represses gene expression to promote cellular proliferation and inhibit differentiation. Here we report the identification and characterization of a cellular protein that antagonizes transcriptional activation and cellular transformation by E1A. This protein, termed CREG for cellular repressor of E1A-stimulated genes, shares limited sequence similarity with E1A and binds both the general transcription factor TBP and the tumor suppressor pRb in vitro. In transfection assays, CREG represses transcription and antagonizes 12SE1A-mediated activation of both the adenovirus E2 and cellular hsp70 promoters. CREG also antagonizes E1A-mediated transformation, as expression of CREG reduces the efficiency with which E1A and the oncogene ras cooperate to transform primary cells. Binding sites for E2F, a key transcriptional regulator of cell cycle progression, were found to be required for repression of the adenovirus E2 promoter by CREG, and CREG was shown to inhibit activation by E2F. Since both the adenovirus E1A protein and transcriptional activation by E2F function to promote cellular proliferation, the results presented here suggest that CREG activity may contribute to the transcriptional control of cell growth and differentiation.

In this issue of Molecular Cell, Molin et al. (2011) reveal that caloric restriction alleviates PKA-dependent inhibition of sulfiredoxin translation, maintaining the thioredoxin peroxidase activity of a peroxiredoxin and increasing the hydrogen peroxide resistance and replicative life span of Saccharomyces cerevisiae.

Increases in hydrogen peroxide can initiate protective responses to limit or repair oxidative damage. However, hydrogen peroxide signals also fine-tune responses to growth factors and cytokines to control cell division, differentiation, and migration. Here we discuss some of the mechanisms by which hydrogen peroxide is sensed and utilized as a signaling molecule to regulate diverse biological processes. We also discuss how the localization and levels of hydrogen peroxide, antioxidants, and the cellular metal composition together influence the nature of the response. Antioxid. Redox Signal. 15, 147–151.

Peroxiredoxins are ubiquitous proteins that are found from bacteria to humans. Until recently they were thought to solely act as antioxidants catalysing the reduction of peroxides through their associated thioredoxin peroxidase activity. However, recent work has begun to uncover hitherto unsuspected roles for one group of these proteins, the typical 2-Cys peroxiredoxins (2-Cys Prx). For example, typical 2-Cys Prxs have been found to have roles in the model organisms Schizosaccharomvces pombe and Saccharomyces cerevisiae in regulating signal transduction, in DNA damage responses and as molecular chaperones. There is increasing evidence that H2O2 is utilised as a signalling molecule to regulate a range of important cellular processes. As abundant and ubiquitous peroxidase enzymes the peroxidase activity of typical 2-Cys Prxs is important in the regulation of these functions. Significantly, studies in yeast suggest that the regulation of the thioredoxin peroxidase and chaperone activities of these multifunction enzymes is an important aspect of H2O2-mediated signal transduction and consequently have provided important insight into the roles of these proteins in higher eukaryotes.

Peroxiredoxins are ubiquitous proteins that are found from bacteria to humans. Until recently they were thought to solely act as antioxidants catalysing the reduction of peroxides through their associated thioredoxin peroxidase activity. However, recent work has begun to uncover hitherto unsuspected roles for one group of these proteins, the typical 2-Cys peroxiredoxins (2-Cys Prx). For example, typical 2-Cys Prxs have been found to have roles in the model organisms Schizosaccharomyces pombe and Saccharomyces cerevisiae in regulating signal transduction, in DNA damage responses and as molecular chaperones. There is increasing evidence that rm H2O2 is utilised as a signalling molecule to regulate a range of important cellular processes. As abundant and ubiquitous peroxidase enzymes the peroxidase activity of typical 2-Cys Prxs is important in the regulation of these functions. Significantly, studies in yeast suggest that the regulation of the thioredoxin peroxidase and chaperone activities of these multi-function enzymes is an important aspect of H2O2–mediated signal transduction and consequently have provided important insight into the roles of these proteins in higher eukaryotes

Although activation of the AP-1-like transcription factor Pap1 in Schizosaccharomyces pombe is important for oxidative stress-induced gene expression, this activation is delayed at higher concentrations of peroxide. Here, we reveal that the 2-Cys peroxiredoxin (2-Cys Prx) Tpx1 is required for the peroxide-induced activation of Pap1. Tpx1, like other eukaryotic 2-Cys Prxs, is highly sensitive to oxidation, which inactivates its thioredoxin peroxidase activity. Our data suggest that the reduced thioredoxin peroxidase-active form of Tpx1 is required for the peroxide-induced oxidation and nuclear accumulation of Pap1. Indeed, in contrast to the previously described role for Tpx1 in the activation of the Sty1 stress-activated protein kinase by peroxide, we find that both catalytic cysteines of Tpx1 are required for Pap1 activation. Moreover, overexpression of the conserved sulfiredoxin Srx1, which interacts with and reduces Tpx1, allows rapid activation of Pap1 at higher concentrations of H(2)O(2). Conversely, loss of Srx1 prevents the reduction of oxidized Tpx1 and prolongs the inhibition of Pap1 activation. Collectively, these data suggest that redox regulation of the thioredoxin peroxidase activity of Tpx1 acts as a molecular switch controlling the transcriptional response to H(2)O(2). Furthermore, they reveal that a single eukaryotic 2-Cys Prx regulates peroxide signaling by multiple independent mechanisms.

We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides. Ybp1 is required for H2O2-induced expression of the antioxidant encoding gene TRX2. Our data indicate that the effects of Ybp1 are mediated through the Yap1 transcription factor. Indeed, Ybp1 forms a stress-induced complex with Yap1 in vivo and stimulates the nuclear accumulation of Yap1 in response to H2O2 but not in response to the thiol-oxidizing agent diamide. The H2O2-induced nuclear accumulation of Yap1 is regulated by the oxidation of specific cysteine residues and is dependent on the thiol peroxidase Gpx3. Our data suggest that Ybp1 is required for the H2O2-induced oxidation of Yap1 and acts in the same pathway as Gpx3. Consequently, Ybp1 represents a novel class of stress regulator of Yap1. These data have important implications for the regulation of protein oxidation and stress responses in eukaryotes.

We have identified three genes, gst1(+), gst2(+), and gst3(+), encoding theta-class glutathione S-transferases (GSTs) in Schizosaccharomyces pombe. The gst1(+) and gst2(+) genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. The gst1Delta, gst2Delta, and gst3Delta mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Both gst2(+) and gst3(+) mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1(+), gst2(+), and gst3(+) are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast.

In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. Here we show that the forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein Fkh1 demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression.

Differentiation of the human embryonal carcinoma cell line NTERA-2 is characterized by changes in morphology, altered patterns of gene expression, reduced proliferative potential, and a loss of tumorigenicity. The cellular repressor of E1A-stimulated genes, CREG, was previously shown to antagonize transcriptional activation and cellular transformation by the Adenovirus E1A oncoprotein. These properties suggested that CREG may function to inhibit cell growth and/or promote differentiation. Here we show that CREG is a secreted glycoprotein which enhances differentiation of NTERA-2 cells. Northern blot analysis reveals that, although CREG mRNA is widely expressed in adult tissues, CREG mRNA is not significantly expressed in pluripotent mouse embryonic stem cells or NTERA-2 embryonal carcinoma cells. CREG mRNA is rapidly induced upon in vitro differentiation of both mouse embryonic stem cells and human NTERA-2 cells. We show that constitutive expression of CREG in NTERA-2 cells enhances neuronal differentiation upon treatment with retinoic acid. Media enriched in CREG was also found to promote NTERA-2 differentiation in the absence of an inducer such as retinoic acid. These studies suggest that secreted CREG protein participates in a signaling cascade important for differentiation of pluripotent stem cells such as those found in teratocarcinomas.

Previous data have indicated the presence of c-myc mRNA and protein in mature skeletal muscle, but whether the protein is present as a Myc/Max heterodimer capable of influencing transcription in that tissue or its potential role in pathological muscle tissue has not been examined. The expression of c-myc in normal and mdx dystrophic mouse skeletal muscle was therefore investigated. C-myc mRNA was detected by Northern hybridisation in normal and dystrophic mouse muscle from mice up to 40 days of age and immunohistochemical staining confirmed that c-myc protein was expressed in muscle fibre nuclei in the muscles of mice up to 40 days of age. The presence of Myc-containing complexes that are able to bind to the concensus Myc/Max binding site was demonstrated in these muscles using the electrophoretic mobility shift assay. These results show that c-myc protein is expressed in functional complexes in both normal and dystrophic mouse skeletal muscle during post-natal maturation. They also show that expression of c-myc in mouse skeletal muscle decreases to undetectable levels by about 40 days of age. Although no differences were detected between the expression of c-myc in mdx and control mouse muscle, these data show that muscle contains Myc protein which has previously been demonstrated to be capable of initiating programmed cell death in other tissues.

Duchenne muscular dystrophy is a sex-linked, progressive muscle wasting disease caused by a defect in the dystrophin gene which results in the absence of dystrophin protein from skeletal muscle. The mdx mouse also has a defect in the dystrophin gene which leads to the absence of dystrophin from skeletal muscle and is therefore used as an animal model of the human disease. In the mdx mouse dystrophin deficiency leads to a multistaged skeletal muscle disorder in which a series of agerelated changes take place. Prior to 14 days of age the muscles of mfx mice show no signs of muscle damage. At around 14 days there commences an acute phase of degeneration with all fibres having undergone at least one cycle of degeneration and regeneration by 40 days of age. Above 40 days of age the amount of muscle degeneration is greatly decreased [I]. The causes of the onset of the acute phase of muscle degeneration are unknown although there is some evidence that it coincides with the maturation of the thyroid [ 1,2]. Degeneration of dystrophic skeletal muscle has generally been accepted to take place via necrosis however recent studies have suggested that the phase of acute muscle degeneration in the mdx mouse occurs via apoptosis [3]. We have examined the expression of two proteins, with fimctions related to programmed cell death, Bcl-2 and Bax, in the muscles of mdx and age-matched control mice. This was done in order to determine whether changes in the levels of either protein could be associated with the onset of muscle degeneration at 14 days of age, or the stabilisation of mdx mouse skeletal muscle above 40 days of age. The expression of Bcl-2 and Bax was examined in 11, 15, 21, 28, 40 day and 1 year old mdx and agematched control mouse muscle by western blotting. 200pg denatured muscle proteins were separated by SDS polyacrylamide electrophoresis and electroblotted onto nitrocellulose membrane. The membranes were blocked then incubated with OSpg/ml rabbit polyclonal anti-Bcl-2 antibodies or 1 .Opg/ml rabbit polyclonal anti-Bax antibodies [Santa Cruz]. The membranes were washed, to remove non-specifically bound primary antibodies, then incubated with peroxidase-conjugated goat polyclonal antirabbit IgG antibodies. The membranes were washed then the secondary antibody was visualised by incubating the membrane with 0.05 % (w/v) diaminobenzidene and 0.02 % (v/v) H,O,. Both Bcl-2 and Bax proteins were expressed in mdx and age-matched control mouse muscle at all the ages examined (see figures 1 and 2). The levels of Bax appeared to decrease with age. There were no differences in the levels of either protein between mdx and control mouse muscle. The only established role of Bcl-2 is in the suppression of programmed cell death. The mechanism by which Bcl-2 inhibits programmed cell death is unknown but appears to require dimerisation with Bax [4]. However, high levels of Bax protein have been shown to accelerate programmed cell death, apparently by preventing Bcl-2 from suppressing programmed cell death PI.