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54-442: GSTs can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyse the conjugation of GSH—via a sulfhydryl group—to electrophilic centers on a wide variety of substrates in order to make the compounds more water-soluble. This activity detoxifies endogenous compounds such as peroxidised lipids and enables the breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins, which gave rise to

108-484: A chain reaction that results in oxidative stress and cell damage . In pathology and medicine , lipid peroxidation plays a role in cell damage which has broadly been implicated in the pathogenesis of various diseases and disease states, including ageing , whereas in food science lipid peroxidation is one of many pathways to rancidity . The chemical reaction of lipid peroxidation consists of three phases: initiation , propagation , and termination . In

162-570: A GSH-binding site. In heterodimeric GST complexes such as those formed by the cytosolic mu and alpha classes, however, the cleft between the two subunits is home to an additional high-affinity nonsubstrate xenobiotic binding site, which may account for the enzymes' ability to form heterodimers. The compounds targeted in this manner by GSTs encompass a diverse range of environmental or otherwise exogenous toxins, including chemotherapeutic agents and other drugs, pesticides, herbicides, carcinogens, and variably-derived epoxides; indeed, GSTs are responsible for

216-567: A form of the GCL protein for the purpose of synthesizing GSH. To further highlight the critical nature of this enzyme, genetic knockdown of GCL results in embryonic lethality. Furthermore, dysregulation of GCL enzymatic function and activity is known to be involved in the vast majority of human diseases, such as diabetes, Parkinson's disease, Alzheimer's disease, COPD, HIV/AIDS, and cancer. This typically involves impaired function leading to decreased GSH biosynthesis, reduced cellular antioxidant capacity, and

270-419: A lipid hydroperoxyl radical ( LOO• ). The lipid hydroperoxyl radical ( LOO• ) can further abstract hydrogen from a new PUFA substrate, forming another lipid radical ( L• ) and now finally a lipid hydroperoxide (LOOH). The lipid hydroperoxyl radical ( LOO• ) can also undergo a variety of reactions to produce new radicals. The additional lipid radical ( L• ) continues the chain reaction , whilst

324-464: A majority of human tumor cell lines and prevalence in chemotherapeutic-resistant tumors, GSTP1-1 is thought to play a role in the development of cancer and its potential resistance to drug treatment. Further evidence for this comes from the knowledge that GSTP can selectively inhibit C -Jun phosphorylation by JNK , preventing apoptosis. During times of low cellular stress, a complex forms through direct protein–protein interactions between GSTP and

378-509: A potential target for diabetic drug treatment. In addition, insulin administration is known to result in increased GST gene expression through the PI3K/AKT/mTOR pathway and reduced intracellular oxidative stress, while glucagon decreases such gene expression. Omega-class GST (GSTO) genes, in particular, are associated with neurological diseases such as Alzheimer's , Parkinson's , and amyotrophic lateral sclerosis ; again, oxidative stress

432-498: A process known as a pull-down assay . This is accomplished by inserting the GST DNA coding sequence next to that which codes for the protein of interest. Thus, after transcription and translation, the GST protein and the protein of interest will be expressed together as a fusion protein . Because the GST protein has a strong binding affinity for GSH, beads coated with the compound can be added to

486-600: A purification tag, GST acts as a chaperone for the attached protein, promoting its correct folding, as well as preventing it from becoming aggregated in inclusion bodies when expressed in bacteria. The GST tag can easily be removed following purification by addition of a protease if a suitable protease-cleavage site has been inserted between the GST-tag and the protein of interest (which is usually included in many commercially available sources of GST-tagged plasmids). Lipid peroxidation Lipid peroxidation , or lipid oxidation ,

540-577: Is a complex chemical process that leads to oxidative degradation of lipids , resulting in the formation of peroxide and hydroperoxide derivatives. It occurs when free radicals , specifically reactive oxygen species (ROS), interact with lipids within cell membranes , typically polyunsaturated fatty acids (PUFAs) as they have carbon–carbon double bonds . This reaction leads to the formation of lipid radicals , collectively referred to as lipid peroxides or lipid oxidation products ( LOPs ), which in turn react with other oxidizing agents , leading to

594-498: Is a growing body of evidence supporting the role of GST, particularly GSTP, in cancer development and chemotherapeutic resistance. The link between GSTP and cancer is most obvious in the overexpression of GSTP in many cancers, but it is also supported by the fact that the transformed phenotype of tumor cells is associated with aberrantly regulated kinase signaling pathways and cellular addiction to overexpressed proteins. That most anti-cancer drugs are poor substrates for GSTP indicates that

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648-423: Is a substrate, isomers of hydroperoxyeicosatetraenoic acid (HPETEs) and hydroxyeicosatetraenoic acids (HETEs) are formed. Antioxidants play a crucial role in mitigating lipid peroxidation by neutralizing free radicals, thereby halting radical chain reactions. Key antioxidants include vitamin C and vitamin E . Additionally, enzymes including superoxide dismutase , catalase , and peroxidase contribute to

702-680: Is believed to be the culprit, with decreased GSTO gene expression resulting in a lowered age of onset for the diseases. The high intracellular concentrations of GSTs coupled with their cell-specific cellular distribution allows them to function as biomarkers for localising and monitoring injury to defined cell types. For example, hepatocytes contain high levels of alpha GST and serum alpha GST has been found to be an indicator of hepatocyte injury in transplantation , toxicity and viral infections. Similarly, in humans, renal proximal tubular cells contain high concentrations of alpha GST, while distal tubular cells contain pi GST. This specific distribution enables

756-452: Is best illustrated by the lethal phenotype of glutathione peroxidase 4 ( GPX4 ) knockout mice. These animals do not survive past embryonic day 8, indicating that the removal of lipid hydroperoxides is essential for mammalian life. It is unclear whether dietary lipid peroxides are bioavailable and play a role in disease, as a healthy human body has protective mechanisms in place against such hazards. Certain diagnostic tests are available for

810-684: Is influenced by numerous factors, including cellular expression of the GCL subunit proteins, access to substrates (cysteine is typically limiting in the production of γ-GC), the degree of negative feedback inhibition by GSH, and functionally relevant post-translational modifications to specific sites on the GCL subunits. Given its status as the rate-limiting enzyme in GSH biosynthesis, changes in GCL activity directly equate to changes in cellular GSH biosynthetic capacity. Therefore, therapeutic strategies to alter GSH production have focused on this enzyme. In keeping with its critical importance in maintaining life, GCL

864-399: Is located in the thioredoxin -like domain of both cytosolic and mitochondrial GSTs. The region containing the greatest amount of variability between the assorted classes is that of helix α2 , where one of three different amino acid residues interacts with the glycine residue of glutathione. Two subgroups of cytosolic GSTs have been characterized based upon their interaction with glutathione:

918-500: Is required to maintain cell viability, expression of the GCL subunits is also inducible in response to oxidative stress , GSH depletion, and exposure to toxic chemicals, with the Nrf2 , AP-1 , and NF-κB transcription factors regulating the inducible and constitutive expression of both subunits In terms of enzyme functional regulation, GSH itself acts as a feedback inhibitor of GCL activity. Under normal physiologic substrate concentrations,

972-473: Is subject to a multi-level regulation of its expression, function, and activity. GCL expression is regulated at the transcriptional (transcription of the GCLC and GCLM DNA to make mRNA), posttranscriptional (the stability of the mRNA over time), translational (processing of the mRNA into protein), and posttranslational levels (involving modifications to the existing proteins). Although baseline constitutive expression

1026-401: Is the first enzyme of the cellular glutathione (GSH) biosynthetic pathway that catalyzes the chemical reaction : L -glutamate + L -cysteine + ATP ⇌ {\displaystyle \rightleftharpoons } γ-glutamyl cysteine + ADP + P i GSH, and by extension GCL, is critical to cell survival. Nearly every eukaryotic cell, from plants to yeast to humans, expresses

1080-465: The C -terminus of JNK, effectively preventing the action of JNK and thus its induction of the JNK pathway. Cellular oxidative stress causes the dissociation of the complex, oligomerization of GSTP, and induction of the JNK pathway, resulting in apoptosis . The connection between GSTP inhibition of the pro-apoptotic JNK pathway and the isozyme's overexpression in drug-resistant tumor cells may itself account for

1134-439: The active site of the enzyme; and subsequently to activate the thiol group of GSH, enabling the nucleophilic attack upon the substrate. The glutathione molecule binds in a cleft between N - and C -terminal domains - the catalytically important residues are proposed to reside in the N -terminal domain. Both subunits of the GST dimer, whether hetero- or homodimeric in nature, contain a single nonsubstrate binding site, as well as

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1188-484: The conjugation products are converted into mercapturic acids and excreted via the urine or bile . Most mammalian isoenzymes have affinity for the substrate 1-chloro-2,4-dinitrobenzene , and spectrophotometric assays utilising this substrate are commonly used to report GST activity. However, some endogenous compounds, e.g., bilirubin, can inhibit the activity of GSTs. In mammals, GST isoforms have cell specific distributions (for example, α-GST in hepatocytes and π-GST in

1242-415: The initiation phase, a pro-oxidant hydroxyl radical ( OH• ) abstracts the hydrogen at the allylic position (–CH 2 –CH=CH 2 ) or methine bridge (=CH−) on the stable lipid substrate, typically a polyunsaturated fatty acid (PUFA), to form the lipid radical ( L• ) and water (H 2 O). In the propagation phase, the lipid radical ( L• ) reacts with molecular oxygen ( O 2 ) to form

1296-399: The nucleophilic attack by GSH on electrophilic carbon, sulfur, or nitrogen atoms of said nonpolar xenobiotic substrates, thereby preventing their interaction with crucial cellular proteins and nucleic acids. Specifically, the function of GSTs in this role is twofold: to bind both the substrate at the enzyme's hydrophobic H -site and GSH at the adjacent, hydrophilic G-site, which together form

1350-429: The oxidation response by reducing the presence of hydrogen peroxide , which is a prevalent precursor of the hydroxyl radical ( OH• ). As an example, vitamin E can donate a hydrogen atom to the lipid hydroperoxyl radical ( LOO• ) to form a vitamin E radical, which further reacts with another lipid hydroperoxyl radical ( LOO• ) forming non-radical products. Phototherapy may cause lipid peroxidation, leading to

1404-404: The GCLC monomer alone may synthesize gamma-glutamylcysteine; however, the normal physiologic levels of GSH (estimated at around 5 mM) far exceeds the GSH K i for GCLC, suggesting that only the GCL holoenzyme is functional under baseline conditions. However, during oxidative stress or toxic insults that can result in the depletion of cellular GSH or its oxidation to glutathione disulfide (GSSG),

1458-606: The Y-GST group, which uses a tyrosine residue to activate glutathione, and the S/C-GST, which instead uses serine or cysteine residues. The porcine pi-class enzyme pGTSP1-1 was the first GST to have its structure determined, and it is representative of other members of the cytosolic GST superfamily, which contain a thioredoxin-like N -terminal domain as well as a C -terminal domain consisting of alpha helices . Mammalian cytosolic GSTs are dimeric , with both subunits being from

1512-546: The alpha, zeta, theta, mu, pi, sigma, and omega classes, while six isozymes belonging to classes I, II, and IV of the MAPEG superfamily are known to exist. Standardized GST nomenclature first proposed in 1992 identifies the species to which the isozyme of interest belongs with a lower-case initial (e.g., "h" for human), which precedes the abbreviation GST. The isozyme class is subsequently identified with an upper-case letter (e.g., "A" for alpha), followed by an Arabic numeral representing

1566-400: The biliary tract of the human liver). GSTs have a role in the bioactivation process of clopidogrel prodrug. Although best known for their ability to conjugate xenobiotics to GSH and thereby detoxify cellular environments, GSTs are also capable of binding nonsubstrate ligands , with important cell signaling implications. Several GST isozymes from various classes have been shown to inhibit

1620-516: The class subfamily (or subunit). Because both mitochondrial and cytosolic GSTs exist as dimers , and only heterodimers form between members of the same class, the second subfamily component of the enzyme dimer is denoted with a hyphen, followed by an additional Arabic numeral. Therefore, if a human glutathione S -transferase is a homodimer in the pi-class subfamily 1, its name will be written as "hGSTP1-1." The early nomenclature for GSTs referred to them as “Y” proteins, referring to their separation in

1674-463: The conjugation of β 1 -8,9-epoxide, a reactive intermediate formed from aflatoxin B 1 , which is a crucial means of protection against the toxin in rodents. The detoxification reactions comprise the first four steps of mercapturic acid synthesis, with the conjugation to GSH serving to make the substrates more soluble and allowing them to be removed from the cell by transporters such as multidrug resistance-associated protein 1 ( MRP1 ). After export,

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1728-562: The dipeptide gamma-glutamylcysteine (γ-GC). This peptide coupling is unique in that it occurs between the amino moiety of the cysteine and the terminal carboxylic acid of the glutamate side chain (hence the name gamma-glutamyl cysteine). This peptide bond is resistant to cleavage by cellular peptidases and requires a specialized enzyme, gamma-glutamyl transpeptidase (γGT), to metabolize γ-GC and GSH into its constituent amino acids. GCL enzymatic activity generally dictates cellular GSH levels and GSH biosynthetic capacity. GCL enzymatic activity

1782-710: The early term for GSTs, ligandin . Protein sequence and structure are important additional classification criteria for the three superfamilies (cytosolic, mitochondrial, and MAPEG) of GSTs: while classes from the cytosolic superfamily of GSTs possess more than 40% sequence homology , those from other classes may have less than 25%. Cytosolic GSTs are divided into 13 classes based upon their structure: alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega. Mitochondrial GSTs are in class kappa. The MAPEG superfamily of microsomal GSTs consists of subgroups designated I-IV, between which amino acid sequences share less than 20% identity. Human cytosolic GSTs belong to

1836-432: The evidence is minimal for the influence of GST polymorphisms of the alpha, mu, pi, and theta classes on susceptibility to various types of cancer, numerous studies have implicated such genotypic variations in asthma , atherosclerosis , allergies , and other inflammatory diseases. Because diabetes is a disease that involves oxidative damage, and GSH metabolism is dysfunctional in diabetic patients, GSTs may represent

1890-412: The evolution of Drosophila GST ​ ​ - which metabolizes both. ​ ​ The activity of GSTs is dependent upon a steady supply of GSH from the synthetic enzymes gamma-glutamylcysteine synthetase and glutathione synthetase , as well as the action of specific transporters to remove conjugates of GSH from the cell. The primary role of GSTs is to detoxify xenobiotics by catalyzing

1944-473: The function of a kinase involved in the MAPK pathway that regulates cell proliferation and death , preventing the kinase from carrying out its role in facilitating the signaling cascade. Cytosolic GSTP1-1, a well-characterized isozyme of the mammalian GST family, is expressed primarily in heart, lung, and brain tissues; in fact, it is the most common GST expressed outside the liver. Based on its overexpression in

1998-541: The function of any monomeric GCLC in the cell is likely to become quite important. In support of this hypothesis, mice lacking expression of the GCLM subunit due to genetic knockdown exhibit low levels of tissue GSH (~10–20% of the normal level), which is roughly the level of the GSH K i for monomeric GCLC. Animal glutamate cysteine ligase (GCL) is a heterodimeric enzyme composed of two protein subunits that are coded by independent genes located on separate chromosomes: In

2052-496: The induction of oxidative stress. However, in cancer, GCL expression and activity is enhanced, which serves to both support the high level of cell proliferation and confer resistance to many chemotherapeutic agents. Glutamate cysteine ligase (GCL) catalyzes the first and rate-limiting step in the production of the cellular antioxidant glutathione (GSH), involving the ATP-dependent condensation of cysteine and glutamate to form

2106-405: The lipid hydroperoxide (LOOH) is the primary end product. The formation of lipid radicals is sensitive to the kinetic isotope effect . Reinforced lipids in the membrane can suppress the chain reaction of lipid peroxidation. The termination step can vary, in both its actual chemical reaction and when it will occur. Lipid peroxidation is a self-propagating chain reaction and will proceed until

2160-422: The lipid substrate is consumed and the last two remaining radicals combine, or a reaction which terminates it occurs. Termination can occur when two lipid hydroperoxyl radicals ( LOO• ) react to form peroxide and oxygen (O 2 ). Termination can also occur when the concentration of radical species is high. The primary products of lipid peroxidation are lipid hydroperoxides (LOOH). When arachidonic acid

2214-436: The majority of cells and tissues, the expression of GCLM protein is lower than GCLC and GCLM is therefore limiting in the formation of the holoenzyme complex. Thus, the sum total of cellular GCL activity is equal to the activity of the holoenzyme + the activity of the remaining monomeric GCLC. composed of a catalytic and a modulatory subunit. The catalytic subunit is necessary and sufficient for all GCL enzymatic activity, whereas

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2268-403: The measurement of urinary GSTs to be used to quantify and localise renal tubular injury in transplantation , nephrotoxicity and ischaemic injury. In rodent pre-clinical studies, urinary and serum alpha GST have been shown to be sensitive and specific indicators of renal proximal tubular and hepatocyte necrosis respectively. GST can be added to a protein of interest to purify it from solution in

2322-416: The modulatory subunit increases the catalytic efficiency of the enzyme. Mice lacking the catalytic subunit (i.e., lacking all de novo GSH synthesis) die before birth. Mice lacking the modulatory subunit demonstrate no obvious phenotype, but exhibit marked decrease in GSH and increased sensitivity to toxic insults. The plant glutamate cysteine ligase is a redox-sensitive homodimeric enzyme , conserved in

2376-490: The plant kingdom. In an oxidizing environment, intermolecular disulfide bridges are formed and the enzyme switches to the dimeric active state. The midpoint potential of the critical cysteine pair is -318 mV. In addition to the redox-dependent control, the plant GCL enzyme is feedback inhibited by glutathione. GCL is exclusively located in plastids , and glutathione synthetase (GS) is dual-targeted to plastids and cytosol, thus GSH and gamma-glutamylcysteine are exported from

2430-564: The plastids. Studies also shown that restricting GCL activity to the cytosol or glutathione biosynthesis to the plastids is sufficient for normal plant development and stress tolerance. Both glutathione biosynthesis enzymes are essential in plants; knock-outs of GCL and GS are lethal to embryo and seedling, respectively. As of late 2007, six structures have been solved for this class of enzymes, with PDB accession codes 1V4G , 1VA6 , 2D32 , 2D33 , 2GWC , and 2GWD . (See Template:Leucine metabolism in humans – this diagram does not include

2484-521: The pro-apoptotic p38 and JNK portions of the MAPK signaling cascade. Like GSTP, GSTM1 interacts with its partner in the absence of oxidative stress, although ASK1 is also involved in heat shock response, which is likewise prevented during ASK1 sequestration. The fact that high levels of GST are associated with resistance to apoptosis induced by a range of substances, including chemotherapeutic agents, supports its putative role in MAPK signaling prevention. There

2538-402: The protein mixture; as a result, the protein of interest attached to the GST will stick to the beads, isolating the protein from the rest of those in solution. The beads are recovered and washed with free GSH to detach the protein of interest from the beads, resulting in a purified protein. This technique can be used to elucidate direct protein–protein interactions. A drawback of this assay is that

2592-560: The protein of interest is attached to GST, altering its native state. A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein. The tag is 220 amino acids (roughly 26 kDa) in size, which, compared to tags such as the Myc-tag or the FLAG-tag , is quite large. It can be fused to either the N -terminus or C -terminus of a protein. In addition to functioning as

2646-559: The quantification of the end-products of lipid peroxidation, to be specific, malondialdehyde (MDA). The most commonly used test is called a TBARS Assay ( thiobarbituric acid reactive substances assay). Thiobarbituric acid reacts with malondialdehyde to yield a fluorescent product. However, there are other sources of malondialdehyde, so this test is not completely specific for lipid peroxidation. Gamma-glutamylcysteine synthetase Glutamate–cysteine ligase (GCL) EC 6.3.2.2 ), previously known as γ-glutamylcysteine synthetase (GCS),

2700-432: The role of elevated GSTP in many tumor cell lines is not to detoxify the compounds, but must have another purpose; this hypothesis is also given credence by the common finding of GSTP overexpression in tumor cell lines that are not drug resistant. In addition to their roles in cancer development and chemotherapeutic drug resistance, GSTs are implicated in a variety of diseases by virtue of their involvement with GSH. Although

2754-581: The rupture of red blood cell cell membranes. End-products of lipid peroxidation may be mutagenic and carcinogenic . For instance, the end-product MDA reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts to them, primarily M 1 G . Reactive aldehydes can also form Michael adducts or Schiff bases with thiol or amine groups in amino acid side chains. Thus, they are able to inactivate sensitive proteins through electrophilic stress. The toxicity of lipid hydroperoxides to animals

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2808-572: The same class of GSTs, although not necessarily identical. The monomers are approximately 25 kDa in size. They are active over a wide variety of substrates with considerable overlap. The following table lists all GST enzymes of each class known to exist in Homo sapiens , as found in the UniProtKB/Swiss-Prot database. Environmental challenge by natural toxins helped to prepare Drosophilae for DDT challenge, ​ ​ ​ by shaping

2862-408: The tumor cells' ability to escape apoptosis mediated by drugs that are not substrates of GSTP. Like GSTP, GSTM1 is involved in regulating apoptotic pathways through direct protein–protein interactions, although it acts on ASK1 , which is upstream of JNK. The mechanism and result are similar to that of GSTP and JNK, in that GSTM1 sequesters ASK1 through complex formation and prevents its induction of

2916-484: The “Y” fraction (as opposed to the “X and Z” fractions) using Sephadex G75 chromatography. As GST sub-units were identified they were referred to as Ya, Yp, etc. with if necessary, a number identifying the monomer isoform (e.g. Yb1). Litwack et al proposed the term “Ligandin” to cover the proteins previously known as “Y” proteins. In clinical chemistry and toxicology, the terms alpha GST, mu GST, and pi GST are most commonly used. The glutathione binding site, or "G-site",

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