Nrf2 and the Keap1/Nrf2, Nrf2/sMaf/ARE Pathways
Bridging the gap between normal redox homeostasis and oxidative stress lies a transcription factor protein known as nuclear factor erythroid 2 (Nrf2).
Nrf2 is a basic leucine zipper (bZIP) protein functioning to regulate the expression of many antioxidant proteins. Nrf2 exists in the cytosol where it is bound to the KEAP1 regulatory molecule. In this condition Nrf2 cannot cross into the nucleus. Under conditions of oxidative stress however, KEAP1 releases its hold on Nrf2 allowing the protein it to translocate into the nucleus where it binds to small Maf proteins and the resulting Nrf2/sMaf heterodimer in turn binds to the Antioxidant Response Element (ARE) on various stress related gene targets. In recent years Nrf2/KEAP1 regulation of the Nrf2/sMAF/ARE transcription pathway has been investigated extensively to assess its possible role in aging and disease. Many studies are showing a positive correlation between Nrf2 nuclear translocation and amelioration of disease resulting in increased interest in Nrf2 promotors and KEAP1 agonists as possible therapeutic agents. Other studies show paradoxical outcomes when considering Nrf2 in relation to cancer. In that case, tumor cells are able to use the Nrf2 system as a means to upregulate antioxidant expression which appears to confer additional chemo-protective properties possibly making the tumor more virulent and resistant to therapy.
Left unchecked, elevated levels of pro-oxidants are capable of affecting changes in lipids, proteins and
DNA, which can be toxic and lead to disease and/or environmental decline. For this reason, oxidative
stress has historically been studied mostly for its negative effects and indeed it has been shown to be
involved with heart, liver, lung and neurologic diseases among others. More recently however, it has
been discovered that brief and controlled levels of oxidative stress are a fundamental component of
many cell signaling processes including many involved in regulating expression and function of key
proteins. This relatively new finding is taking the study of oxidative stress in new directions, wherein
researchers believe that oxidative regulation of protein function and the genetic mechanisms of
oxidative stress response may hold the keys to ameliorating the effects of aging and disease.
Among the key stress response elements known to be regulated by Nrf2 include:
The NWLSS™ Catalase activity assay method is essentially that described by Beers and Sizer, 1952 in which the decomposition of peroxide is monitored at 240 nm, with modifications to increase robustness and convenience. Our method uses a certified standard with known catalase activity units so that calibration of precise H2O2 concentration is not necessary in our assay. Similarly, experiments can be carried out at room temperature under conditions that are more accurate and convenient. Modifications are also made in our formulations to overcome problems associated with instability of diluted hydrogen peroxide and diluted enzyme standards at the room temperature so there is no need to keep them on ice and no time wasted to bring them to the assay temperature before each individual assay.
Glutathione (Precursors): Glutamate-cysteine ligase (gamma-glutamylcysteine synthetase)
Glutathione (GSH, g-glutamylcysteinylglycine), the primary non-protein sulfhydryl in aerobic organisms is synthesized in most cells. The ubiquitous tripeptide is formed by the combination of glutamic acid and cysteine, catalyzed by g-glutamylcysteinyl synthetase. Glycine is then added by glutathione synthetase to form GSH.
Glutathione Peroxidase (GPx) (EC 18.104.22.168) enzymes belong to a family of selenoproteins whose function is to catalyze the reduction of various peroxides. Four types of GPx (GPx-1 to 4) have been identified in mammalian species. GPx-1 is the classic intracellular form while GPx-2 to 4 are gastrointestinal, plasma and phospholipid hydroperoxide metabolizing forms respectively.
Glutathione Reductase is a ubiquitous 100-120 kD dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using b-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor. A measurable fraction of GR derived from various sources is often found as the inactive apoenzyme. Dietary supplementation of riboflavin or thyroxin has been shown to activate GR. The in vitro addition of FAD is the basis of assessing riboflavin deficiency. The active site for GR contains a flavin adenine dinucleotide (FAD) and a disulfide. In the presence of NADPH, there is a two electron reduction of GR to produce a semiquinone of FAD, a sulfur radical and a thiol. Purified GR tends to form aggregates in the absence of thiols and these aggregates retain full enzymatic activity. Purified enzyme is reversibly inactivated by NADPH, m-nicotinamide adenine dinucleotide (NADH), GSH, dithionite or borohydride. This inactivation likely requires the presence of divalent cations such as Zn++ and Cd++. The GR enzyme is fully protected from inactivation by ethylenediamine tetraacetic acid (EDTA). Inactivated GR is activated by GSSG, NADP+ and NAD+. In vivo, GR activity is regulated through a redox interconversion mechanism mediated by GSSG regulation of the NADPH generating pathways.
Glutathione S-Transferase (GST) has multiple isoforms; This assay is specific for Glutathione S Transferase Alpha (GSTA) and is not known to cross react with the mu, pi, or theta variants. GSTA is a common biomarker for hepatocellular damage. It also conjugates GSH to 4-hydroxynonenal, a product of lipid peroxidation and is an important player in cellular antioxidant defense mechanisms.