Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Regulation of reactive oxygen species in stem cells and cancer stem cells

Stem cells are defined by their ability to self-renew and their multi-potent differentiation capacity. As such, stem cells maintain tissue homeostasis throughout the life of a multicellular organism. Aerobic metabolism, while enabling efficient energy production, also generates reactive oxygen species (ROS), which damage cellular components. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer, and various anti-oxidative and anti-stress mechanisms of stem cells have been characterized. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, i.e., regulation of the cell cycle. An active area of current research is redox regulation in various cancer stem cells, the malignant counterparts of normal stem cells that are viewed as good targets of cancer therapy. In contrast to cancer cells, in which ROS levels are increased, some cancer stem cells maintain low ROS levels, exhibiting redox patterns that are similar to the corresponding normal stem cell. To fully elucidate the mechanisms involved in stem cell maintenance and to effectively target cancer stem cells, it is essential to understand ROS regulatory mechanisms in these different cell types. Here, the mechanisms of redox regulation in normal stem cells, cancer cells, and cancer stem cells are reviewed.     

Single cystathionine β-synthase domain-containing proteins modulate development by regulating the thioredoxin system in Arabidopsis

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.     

Oxidative activation of antioxidant defence

Living cells maintain a delicate balance between oxidizing and reducing species, and many disorders such as rheumatoid arthritis and Alzheimer's disease have been associated with a disturbed intracellular 'redox equilibrium'. The past few years have witnessed accelerated research into how natural redox responses and antioxidant defence systems are activated and how they restore a healthy redox balance. To function properly, many of these processes rely on a powerful sulfur redox chemistry, which is best exemplified by the complex, newly emerging cysteine-based redox regulation of the glutathione and thioredoxin pathways. Other redox systems based on oxidatively activated amino acid side chains in proteins are also becoming increasingly important, but are still barely understood or explored.     

Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress

To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.     

New equations for redox and nano-signal transduction

Cells maintain redox potentials (Eh) in intracellular compartments, sometimes referred to as redox environments. These potentials are often very reducing, for example in the cytoplasm, but throughout the cell different potentials are maintained, commensurate with the functionality of that particular part of the cell. Furthermore, within a simple cellular compartment, "hot-spots" of redox poise may be maintained. However, despite this complexity, the quantification of such redox potentials has been attempted, and there is indeed a need to accurately assess such potentials, and to monitor how they might change with time. Changes in intracellular potentials may control the oxidation or reduction of protein residues, such as cysteine, which would alter the conformation of those proteins and so modulate their function. Although there are several methods for estimating the intracellular redox potential, the most accessible technique is the measurement of intracellular concentrations of GSH and GSSG, and the calculation of Eh using the Nernst equation. However, using this equation shows that the Eh imposed by the glutathione couple is dependent on the total concentration of glutathione present, and therefore values of Eh obtained may be erroneous. Here, we suggest new equations that can be used to calculate the redox environments of cells.     

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple

Redox state is a term used widely in the research field of free radicals and oxidative stress. Unfortunately, it is used as a general term referring to relative changes that are not well defined or quantitated. In this review we provide a definition for the redox environment of biological fluids, cell organelles, cells, or tissue. We illustrate how the reduction potential of various redox couples can be estimated with the Nernst equation and show how pH and the concentrations of the species comprising different redox couples influence the reduction potential. We discuss how the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an important indicator of redox environment. There are many redox couples in a cell that work together to maintain the redox environment; the GSSG/2GSH couple is the most abundant redox couple in a cell. Changes of the half-cell reduction potential (E(hc)) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation E(hc) approximately -240 mV; differentiation E(hc) approximately -200 mV; or apoptosis E(hc) approximately -170 mV. These estimates can be used to more fully understand the redox biochemistry that results from oxidative stress. These are the first steps toward a new quantitative biology, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.     

The redox-associated adaptive response of brain to physical exercise

Abstract

Reactive oxygen species (ROS) are continuously generated during metabolism. ROS are involved in redox signaling, but in significant concentrations they can greatly elevate oxidative damage leading to neurodegeneration. Because of the enhanced sensitivity of brain to ROS, it is especially important to maintain a normal redox state in brain and spinal cord cell types. The complex effects of exercise benefit brain function, including functional enhancement as well as its preventive and therapeutic roles. Exercise can induce neurogenesis via neurotrophic factors, increase capillarization, decrease oxidative damage, and enhance repair of oxidative damage. Exercise is also effective in attenuating age-associated loss in brain function, which suggests that physical activity-related complex metabolic and redox changes are important for a healthy neural system.     

The Reducible Disulfide Proteome of Synaptosomes Supports a Role for Reversible Oxidations of Protein Thiols in the Maintenance of Neuronal Redox Homeostasis

Neurochemical Research - The mechanisms by which neurons maintain redox homeostasis, disruption of which is linked to disease, are not well known. Hydrogen peroxide, a major cellular oxidant and...     

Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.     

Redox-shuttling between chloroplast and cytosol: integration of intra-chloroplast and extra-chloroplast metabolism

Reducing equivalents produced in the chloroplast are essential for many key cellular metabolic enzyme reactions. Two redox shuttle systems transfer reductant out of the chloroplast; these systems consist of metabolite transporters, coupled with stromal and cytosolic dehydrogenase isozymes. The transporters function in the redox shuttle and also operate as key enzymes in carbon/nitrogen metabolism. To maintain adequate levels of reductant and proper metabolic balance, the shuttle systems are finely controlled. Also, in the leaves of C(4) plants, cell-specific division of carbon and nitrogen assimilation includes cell-specific localization of the redox shuttle systems. The redox shuttle systems are tightly linked to cellular metabolic pathways and are essential for maintaining metabolic balance between energy and reducing equivalents.     

Redox-based endoplasmic reticulum dysfunction in neurological diseases

The redox homeostasis of the endoplasmic reticulum lumen is characteristically different from that of the other subcellular compartments. The concerted action of membrane transport processes and oxidoreductase enzymes maintain the oxidized state of the thiol-disulfide and the reducing state of the pyridine nucleotide redox systems, which are prerequisites for the normal functions of the organelle. The powerful thiol-oxidizing machinery allows oxidative protein folding but continuously challenges the local antioxidant defense. Alterations of the cellular redox environment either in oxidizing or reducing direction affect protein processing and may induce endoplasmic reticulum stress and unfolded protein response. The activated signaling pathways attempt to restore the balance between protein loading and processing and induce apoptosis if the attempt fails. Recent findings strongly support the involvement of this mechanism in brain ischemia, neuronal degenerative diseases and traumatic injury. The redox changes in the endoplasmic reticulum are integral parts of the pathomechanism of neurological diseases, either as causative agents, or as complications.     

Development of redox potential-controlled schemes for very-high-gravity ethanol fermentation

Fermentation redox potential reflects the momentary physiological status of organisms. Controlling redox potential can modulate the redistribution of intracellular metabolic flux to favor the formation of the desired metabolite. Accordingly, we have developed three redox potential-controlled schemes to maximize their effects on the very-high-gravity (VHG) ethanol fermentation. They are aeration-controlled scheme (ACS), glucose-controlled feeding scheme (GCFS), and combined chemostat and aeration-controlled scheme (CCACS). These schemes can maintain fermentation redox potential at a prescribed level (i.e., -50, -100, and -150 mV) by supplementing sterile air, fresh glucose media, or a combination of sterile air and fresh glucose media into a fermenter to counteract the decline of redox potential due to yeast growth. When ACS was employed, the fermentation efficiency at -150 mV is superior to the other two redox potential levels especially when the initial glucose concentration is higher than 250 g/l. The redox potential-controlled period for ACS, GCFS, and CCACS at -150 mV under the same 200 g glucose/l condition was 2.5, 21.7 and 64.6h and the corresponding fermentation efficiency was 85.9,89.3 and 92.7%, respectively.     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

Cellular metabolic and autophagic pathways: Traffic control by redox signaling

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy–lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function.