Глутатион S-трансферазалар отбасысы және олардың функциялары

Family of enzymes

Glutathione S transferases (GSTs), previously known as ligandins, are a family of eukaryotic and prokaryotic phase II metabolic isozymes best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. The GST family consists of three superfamilies: the cytosolic, mitochondrial, and microsomal—also known as MAPEG—proteins. Members of the GST superfamily are extremely diverse in amino acid sequence, and a large fraction of the sequences deposited in public databases are of unknown function. 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.

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 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.

Environmental challenge by natural toxins helped to prepare Drosophilae for DDT challenge, 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 the active site of the enzyme; and subsequently to activate the thiol group of GSH, enabling the nucleophilic attack upon the substrate. Both subunits of the GST dimer, whether hetero or homodimeric in nature, contain a single nonsubstrate binding site, as well as 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 the conjugation of β1 8,9 epoxide, a reactive intermediate formed from aflatoxin B1, which is a crucial means of protection against the toxin in rodents. The detoxification reactions comprise the first four steps of mercapturic acid synthesis, 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 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 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 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. 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 the tumor cells' ability to escape apoptosis mediated by drugs that are not substrates of GSTP.

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 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. 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 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 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 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 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 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 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 thrombin protease if a suitable 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).