Glutathione S -transferases ( GSTs ), formerly known as ligandins , comprises the eukaryotic and procaryotic family of phase II isozymes metabolites are notorious for their ability to catalyze the conjugation of reduced-glutathione (GSH) forms into xenobiotic substrates for detoxification purposes. The GST family consists of three superfamilies: cytosol, mitochondria, and microsomal - also known as MAPEG - proteins. Members of the GST superfamily are very diverse in the amino acid sequence, and most of the sequences stored in public databases are unknown functions. The Enzyme Function Initiative (EFI) uses GST as a superfamily model to identify new GST functions.
GST can form up to 10% of the cytosolic protein in some mammalian organs. GST catalyzes GSH conjugation - via sulfhydryl groups - to the electrophilic center on a wide variety of substrates to make the compound more soluble in water. This activity detoxifies endogenous compounds such as lipid peroxidation and enables xenobiotic breakdown. GST can also bind toxins and function as a transport protein, which gives rise to the initial term for GST, ligandin.
Video Glutathione S-transferase
Classification
The sequence and structure of proteins are important additional classification criteria for the three superfamilies (cytosol, mitochondria, and MAPEG) of GST: while the class of GOC cytosolic superfamily has more than 40% homology sequence, those from other classes may have less than 25%. GST cytosol is divided into 13 classes based on its structure: alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega. Mitochondrial GST is in kappa class. MAPEG superfamily GST microsomal consists of a subgroup designated I-IV, in which the amino acid sequence shares less than 20% identity. Human cytosolic GST belongs 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.
Nomenclature
The standard GST nomenclature, first proposed in 1992, identifies the species whose isozymes are in demand by the lowercase initials (eg, "h" for humans), which precede the GST abbreviation. The isozymic class is then identified by capital letters (eg, "A" for alpha), followed by Arabic numerals representing the subfamily class (or subunit). Since both GST mitochondria and cytosol exist as dimers, and only heterodimers are formed between members of the same class, the second subfamily component of the enzyme dimer is denoted by a hyphen, followed by additional Arabic numerals. Therefore, if human glutathione is S -transferase is a homodimer in the subfamily of class pi 1, its name will be written as "hGSTP1-1."
The initial nomenclature for GST refers to them as the "Y" protein, referring to their separation in the fraction "Y" (compared to the fractions "X and Z") using Sephadex G75 chromatography. As the GST sub-units are identified they are called Yes, Yp, etc. with if necessary, a number that identifies the monomer isoform (eg Yb1). Litwack et al proposed the term "Ligandin" to cover the protein previously known as "Y" protein.
In clinical chemistry and toxicology, the term alpha GST, mu GST, and pi GST are most commonly used.
Maps Glutathione S-transferase
Structure
The glutathione binding site, or "G-site," lies within domains such as thioredoxin from both GST cytosol and mitochondria. The region that contains the greatest amount of variability among the various classes is the helix? 2, where one of three different amino acid residues interacts with glycine glutathione residues. Two subgroups of GST cytosol have been characterized based on their interactions with glutathione: the Y-GST group, which uses tyrosine residues to activate glutathione, and S/C-GST, which instead use serine or cysteine ​​residues.
"GST proteins are globular proteins with helical domains and beta-strands of N-terminal mixtures and C-terminal all-helical domains."
Porcine pi-class enzyme pGTSP1-1 is the first GST to have a defined structure, and is representative of other members of the GC cytosolic superfamily, which contains the N-terminal thioredoxin domain as well as the C-terminal domain. consists of an alpha helix.
GST cytosol mammals are dimerik, with both subunits originating from the same GST class, although not necessarily identical. The monomer is about 25 kDa. They are active on a variety of substrates with considerable overlap. The following table lists all GST enzymes from each class that are known to exist in Homo sapiens , as found in the UniProtKB/Swiss-Prot database.
Function
GST activity depends on the steady supply of GSH from synthetic gamma-glutamylcysteine ​​synthetase synthetase and glutathione synthetase, as well as the action of special transporters to remove GSH conjugate from cells. The main role of GST is to detoxify xenobiotics by catalyzing nucleophilic attacks by GSH on carbon, sulfur, or electrophilic nitrogen from nonpolar xenobiotic substrates, thus preventing their interaction with important cellular proteins and nucleic acids. Specifically, the function of GST in this role is twofold: to bind both substrates in the enzyme hydrophobic H-site and GSH in adjacent, hydrophilic G-sites, which together form the active site of the enzyme; and then to activate the THiol GSH group, allowing nucleophilic attacks on the substrate. The glutathione molecule binds in the gap between the N and C-terminal domains - a catalytically important residue is proposed to be 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 a GSH binding site. In heterodimeric GST complexes such as those formed by your classes and alpha cytosolic, however, the gap between the two subunits is home to an additional high affinity xenobiotic binding site, which may explain the enzyme's ability to form heterodimers.
Compounds targeted in this manner by GSTs include a variety of environmental or otherwise exogenous toxins, including chemotherapy agents and other drugs, different pesticides, herbicides, carcinogens and epoxides; indeed, GST is responsible for conjugation? 1 -8,9-epoxide, a reactive intermediate formed from aflatoxin B 1 , which is an important means of protecting toxins in rodents. The detoxification reaction consists of the first four steps of Merkapturic acid synthesis, with conjugation to GSH which serves to make the substrate more soluble and allows them to be removed from cells by transporters such as proteins associated with double resistance 1 (MRP1). After export, the conjugate product is converted to mercaptic acid and excreted through urine or bile.
Most mammalian isoenzymes have an affinity for the 1-chloro-2,4-dinitrobenzene substrate, and the spectrophotometric test utilizing this substrate is usually used to report GST activity. However, some endogenous compounds, for example, bilirubin, may inhibit GST activity. In mammals, GST isoforms have a specific distribution of cells (eg, alpha GST in hepatocytes and pi GST in human liver bile ducts).
Roles in cell signaling
Although well known for its ability to conjugate xenobiotics to GSH and thus detoxify the cellular environment, GST is 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 kinases involved in MAPK pathways that regulate cell proliferation and death, preventing kinases from performing their role in facilitating signaling cascades.
Cytosolic GSTP1-1, a well-characterized isozyme of the mammalian GST family, is expressed primarily in the heart, lungs, and brain tissue; in fact, it is the most common GST that is expressed outside the heart. Based on overexpression in most human tumor cell lines and the prevalence of chemotherapy-resistant tumors, GSTP1-1 is considered to play a role in cancer progression and 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 periods of low cellular stress, a complex form through the interaction of direct proteins between GSTP and C-terminus JNK, effectively prevents the action of JNK and thus induces the JNK pathway. Cellular oxidative stress causes complex dissociation, GSTP oligomerization, and JNK path induction, resulting in apoptosis. The association between GSTP inhibition of pro-apoptotic JNK pathways and isozyme overexpression in drug-resistant tumor cells may in itself explain the ability of tumor cells to escape drug-mediated apoptosis that is not a GSTP substrate.
Like GSTP, GSTM1 is involved in regulating apoptotic pathways through direct protein-protein interactions, although acting on ASK1, which is upstream of JNK. The mechanism and results are similar to GSTP and JNK, where GSTM1 alienates ASK1 through complex formations and prevents the induction of p38 pro-apoptosis and the JNK portion of the MAPK signaling cascade. Like GSTP, GSTM1 interacts with its counterpart in the absence of oxidative stress, although ASK1 is also involved in the heat shock response, which is also prevented during ASK1 sequestration. The fact that high GST levels are associated with resistance to apoptosis caused by various substances, including chemotherapy agents, supports its putative role in the prevention of MAPK signals.
Implications on cancer development
There is growing evidence supporting the role of GST, especially GSTP, in cancer development and chemotherapy resistance. The association between GSTP and cancer is most evident in GSTP overexpression in many cancers, but is also supported by the fact that the tumor phenotype of altered tumors is associated with unusually regulated kinase signal pathways and cellular addiction to excessively overexpressed proteins. That most anti-cancer drugs are a bad substrate for GSTP suggests that the increasing role of GSTP in many tumor cell lines is not to detoxify the compound, but must have other objectives; This hypothesis is also given belief by the general findings of GSTP overexpression in non-drug-resistant tumor cell lines.
Clinical interests
In addition to their role in the development of cancer and chemotherapy drug resistance, GST is involved in various diseases based on their involvement with GSH. Although minimal evidence for the influence of GST polymorphisms from alpha, mu, pi, and theta classes on susceptibility to many types of cancer, many studies have implicated variations in genotypes such as asthma, atherosclerosis, allergies, and other inflammatory diseases.
Because diabetes is a disease that involves oxidative damage, and dysfunctional GSH metabolism in diabetic patients, GST may represent potential targets for the treatment of diabetes drugs. In addition, insulin administration is known to result in increased GST gene expression via the PI3K/AKT/mTOR pathway and decrease intracellular oxidative stress, while glucagon decreases the expression of the gene.
GST Omega-class GST (GSTO), in particular, is 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 expression of the GSTO gene resulting in a lower onset age for the disease.
Release of GST as an indication of organ damage
High intracellular GSTs concentration coupled with cell-specific cellular distributions enables them to function as biological markers to localize and monitor injury to specified cell types. For example, hepatocytes contain high alpha GST levels and serum alpha GST has been found to be an indicator of hepatocyte injury in transplantation, toxicity and viral infection.
Similarly, in humans, the renal proximal tubular cells contain a high concentration of GST alpha, while the distal tubular cells contain pi GST. This specific distribution allows measurement of urinary GSTs used to measure and localize renal tubular injury in transplantation, nephrotoxicity, and ischemic injury.
In pre-clinical studies of rodents, urine GST and alpha serum have proven to be sensitive and specific tubular tubular proximal tubular indicators and hepatocytes respectively.
GST can be added to the desired protein to purify it from a solution in a process known as a pull-down test. This is done by entering the sequence of GST DNA encoding next to the code for the desired protein. Thus, after transcription and translation, the desired GST and protein proteins will be expressed together as fusion proteins. Since GST proteins have a strong binding affinity for GSH, the beads coated with the compound can be added to the protein mixture; as a result, the proteins attached to the GST will attach to the beads, isolating the protein from the rest in the solution. The beads are found and washed with free GSH to release the attractive protein from the beads, producing a purified protein. This technique can be used to explain the interactions of direct proteins. The downside of this test is that the desired protein is attached to the GST, changing its original state.
A GST tag is often used to separate and purify proteins containing GST-fusion proteins. The tag is 220 amino acids (approx. 26 KDa) in size, which, compared to tags like Tag-Myc or the FLAG tag, is quite large. It can blend with N-terminus or C-terminus protein. However, many commercially available sources of GST-tagged plasmids include thrombin domains for cleavage of GST tags during protein purification.
See also
- Affinity chromatography
- Bacterial glutathione transferase
- Glutathione S-transferase Mu 1
- Glutathione S-transferase, C-terminal domain
- GSTP1
- Maltose binding protein
- The protein tag
References
External links
- Overview of Glutathione-S-Transferases
- UMR Orientation Protein in Membrane family/superfamily-199 - MAPEG protein (Eicosanoid and Glutathione metabolism) family
- Glutathione S-Transferase at US National Library of Medicine's Medical Subject Headings (MeSH)
- EC 2.5.1.18
- GST Fusion Protein Preparation
- GST Fusion Fusion System Handbook
Source of the article : Wikipedia
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