Advances in mammalian glutathione transferase research

 1 Discovery of glutathione transferase

The research on glutathione transferase can be traced back to 1961, when Booth et al.[1] found an enzyme catalyzing the reaction between GSH and 2-chloro-4-nitrobenzene in mouse liver cell extracts; in 1978, Lawrence et al.[2] determined that there existed a selenium-free glutathione peroxidase in mouse liver tissues, which was named glutathione transferase, and it marked the first mammalian glutathione transferase gene discovery. This marked the discovery of the first mammalian glutathione peroxidase gene and opened the way for the molecular biology of glutathione peroxidase.

 

2 Classification and nomenclature of glutathione transferases

Mammalian glutathione transferase enzymes are currently classified into three groups: soluble cytosolic, mitochondrial and microsomal glutathione transferase. Soluble cytoplasmic glutathione transferase is a homo- or heterodimer of two subunits, each with a molecular weight of about 23 kDa 30 kDa and consisting of 199 to 244 amino acids. Different combinations of subunits form multiple isozymes, which greatly increases the diversity of glutathione transferase in mammals. According to existing studies, mammals have 15-20 soluble cytoplasmic glutathione transferase[3] . Although there is no uniform standard for the classification of glutathione transferases, mammalian soluble cytoplasmic glutathione transferases have been classified as alpha(α), mu(μ), pi(π), theta(θ), sigma(σ), omega(ω), and zeta(ζ) based on chromosomal location, subunit structure, amino acid sequence similarity, and enzymatic properties. The sequence similarity of the same class is more than 40%, while the sequence similarity between classes is less than 25%.

 

Each subunit of soluble cytoplasmic glutathione transferase is encoded by a separate gene. For example, α, μ, π, θ, σ, ω, and ζ are encoded by glutathione transferase A, glutathione transferase M, glutathione transferase P, glutathione transferase T, glutathione transferase S, and glutathione transferase Z, each of which consists of 7, 8, 7, 5, 5, 5, 5, and 8 exons, respectively.  With the development of genome sequencing technology and bioinformatics tools, more and more gene annotations and chromosome maps have been constructed, revealing the genomic distribution of glutathione transferases in organisms.

 

In humans, for example, the human α-glutathione transferase isoform is encoded by five functional genes (glutathione transferase A1-5) and seven pseudogenes (glutathione transferase AP1-7) clustered together on chromosome 6.  The μ-glutathione transferase contains five members encoded by the glutathione transferase M1-5 gene on chromosome 1, suggesting that the glutathione transferase genes evolved by gene duplication to form different glutathione transferase s. The π, θ, σ, ω, and ζ-glutathione transferase enzymes are located on chromosomes 11, 22, 4, 10, and 14 and are encoded by the 1 (glutathione transferase P1), 3 (glutathione transferase AP1), 3 (glutathione transferase AP1-7), 3 (glutathione transferase AP1-7), and 7 pseudogenes (glutathione transferase AP1-7), which are clustered together and located on chromosome 6. It is coded by 1 (glutathione transferase P1), 3 (glutathione transferase T1, glutathione transferase T2, glutathione transferase T2B), 1 (glutathione transferase S1), 2 (glutathione transferase O1-2), and 1 (glutathione transferase Z1) motifs [4]. With the discovery of more and more glutathione transferase isozymes, in order to avoid confusion, a unified naming system for mammalian glutathione transferase s has been adopted[5] , including the species name prefix, glutathione transferase type and subunit type, etc., e.g.: Hsa glutathione transferase M1 indicates that the gene encodes the 1-subunit of the human μ-glutathione transferase.

 

Mitochondrial glutathione transferase contains only Kappa-like isozymes and is a dimeric protein consisting of 226 amino acids encoded by the glutathione transferase K1 gene with 8 exons located on human chromosome 7 and a single copy in mammals[6] .

 

Microsomal glutathione transferase is a special subfamily of the glutathione transferase superprotein family, with low sequence similarity (<10%) to soluble glutathione transferase, about 150 amino acids, containing four classes of members, I, II, III and IV, of which isozymes I, II, and IV are found in mammals and are closely related to arachidonate formation, and are therefore also known as membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG). The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are closely related to the formation of arachidonic acid, so they are also known as arachidonic acid-like membrane associated proteins (MAPEG). There are six MAPEG genes in humans, and based on sequence similarity, M glutathione transferase 2, leukotriene C4 synthase (LTC4S) and 5-lipoxygenase-activating protein (FLAP) are classified as class I; M glutathione transferase 1 and prostaglandin E2 are classified as class II; and MAPEG is classified as class III. M glutathione transferase 1 and prostaglandin E2 synthase 1 (PGES1) are classified as class IV; class II contains only M glutathione transferase 3. The subunit composition of microsomal glutathione transferase is relatively diversified, such as: M glutathione transferase 1, LTC4S and PGES1 mainly exist in the form of homotrimers[7-9] , while FLAP can form monomers, monomers, homodimers, and homodimers[11] , while FLAP can form monomers, monomers, and monomers. M glutathione transferase 1, LTC4S and PGES1 exist mainly as homotrimers[7-9] , while FLAP can form monomers, dimers and trimers[10] .

 

3 Glutathione transferase Structural characterization and enzymatic reaction

Although there is a great deal of sequence variability among the different types of glutathione transferase, the secondary and higher structures are very similar. In contrast, mitochondrial glutathione transferase adds a helical domain to the βα β motif to bind electrophilic substrates, suggesting a parallel evolution of the crystal structures of soluble cytoplasmic and mitochondrial glutathione transferase[11] . The entire N-terminal domain of soluble glutathione transferase is thought to have evolved from the folded structures of the thioredoxin superfamily, such as thioredoxin and glutaredoxin.

 

Each subunit of glutathione transferase has its own catalytic site of glutathione transferase: structural domain I mainly provides the GSH binding site, called the G site, which is highly conserved in mammals, e.g., tyrosine (Tyr) at position 7 and serine (Ser) at position 17 in alpha/mu/pi-glutathione transferase; structural domain II is responsible for binding hydrophobic substrates, i.e., H site, which is structurally more variable[12-13] . Domain II is responsible for binding the hydrophobic substrate, i.e., the H site, and has a high degree of structural variability[12-13] . In the catalytic reaction, the binding of glutathione transferase to GSH follows the "fit-inducing mechanism" of enzyme and substrate, i.e., the binding of the substrate to the enzyme induces a corresponding change in the conformation of the enzyme protein, which results in the formation of an enzyme-substrate complex by the binding of the enzyme to the substrate and causes the reaction of the substrate; after the reaction is completed and the product is detached from the enzyme, the enzyme is not able to bind to the substrate. When the reaction is completed and the product is detached from the enzyme, the enzyme's activity center returns to its original conformation. The conserved G site promotes the ionization of the hydroxyl group of tyrosine/serine by hydrogen bonding with the thiol group of GSH, resulting in the production of thiolate anions, while the variable H site, when bound to a hydrophobic substrate (electrophilic compounds), participates in a series of reactions driven by the thiolate anion[14] . Mitochondrial glutathione transferase and soluble glutathione transferase have similar catalytic functions.

 

Studies have shown that microsomal glutathione transferase has 3~4 transmembrane domains, the amino and carboxyl termini of the protein protrude into the lumenal side of the membrane, and the GSH-substrate binding site may be located in the cytoplasmic lysate-facing loop[15- 17] . The above results are only structural predictions for microsomal glutathione transferase, and the detailed protein crystal structure of microsomal glutathione transferase needs to be further investigated.

 

4 Expression and functional significance of glutathione transferases

With the development of life sciences and the expansion of research fields, more and more new genes have been gradually discovered, and the determination of the function and genetic information of new genes has become a very important content, therefore, the investigation of the function of mammalian glutathione transferase has also become a hot point of concern for scientists. Glutathione transferase, as an important component in the detoxification system of the body, is responsible for catalyzing the binding of GSH to a variety of electrophilic exogenous compounds (e.g. drugs, industrial intermediates, pesticides, herbicides, environmental pollutants, carcinogens, etc.), and transferring glutathione to the body under the action of multidrug resistance-associated proteins (MRPs). Under the action of multidrug resistance-associated proteins (MRPs), glutathione conjugates are excreted from the body, thus preventing electrophilic reagents from covalently binding to cellular biomolecules during biotransformation, and playing a detoxification role[3] .

 

Although the members of the glutathione transferase family originated from the same ancestor, with the accumulation of gene duplication, recombination and mutation, different classes of glutathione transferase exhibit substrate specificity and functional diversity in their catalytic activity. For example, the alpha members of glutathione transferase A1-4, which are expressed in human liver and kidney, have different substrate binding properties despite their highly homologous sequences and protein structures. Glutathione transferase A1, glutathione transferase A2 and glutathione transferase A3 tend to bind 2,4-dinitrochlorobenzene (CDNB), and their catalytic activities are significantly higher than that of allyl aldehydes. On the contrary, glutathione transferase A4 prefers allyl aldehydes, and its catalytic activity for allyl aldehydes is nearly 200 times higher than that of glutathione transferase A1[18] . It has been shown that glutathione transferase A1 and glutathione transferase A4 have specific substrate binding pockets located in the α1-β1 loop and in the C-terminal α4 helix region, and the assembly and position of amino acids in this region determine the shape and characteristics of the substrate binding pockets, and thus the specificity of substrate recognition of the two enzymes[18] . In addition, Björnestedt et al. reported that the Arg15 and Tyr9 sites of glutathione transferase A1 are important in binding and activating GSH to maintain enzyme activity [19]. Further studies have also revealed a number of key sites affecting the catalytic activity of glutathione transferase A4 towards allyl aldehydes, including Gly12 in the α1-β1 loop, Ile107 (Leu), Met108 (Leu), and Phe111 (Val) in the α4 helix region, as well as the C-terminal sites of Pro208 (Met), Tyr212 (Ser), Val213 (Leu) and Phe111 (Val) in the α1-β1 loop. (Leu), Val 216 (Ala) and Pro222 (Phe) at the C-terminus[18-19] , which have contributed to the understanding of their catalytic mechanisms.

 

In addition, glutathione transferase also has non-catalytic functions, for example, glutathione transferase O1 regulates the lanylate receptor, an endoplasmic reticulum calcium ionophore protein, and prevents apoptosis by inhibiting its activity[20] . pi-glutathione transferase is mainly expressed in the placenta, erythrocytes, breast, lung and prostate. pi-glutathione transferase is an inhibitor of C-Jun amino-terminal kinase 1 (JNK1), which regulates apoptosis, stress and cell proliferation by inhibiting the activity of JNK, a subclass of the MAP kinase signaling pathway. Mu-glutathione transferase M1 is an endogenous inhibitor of apoptosis signal-regulating kinase 1 (ASK1), which regulates apoptosis induced by stress or cytokines by inhibiting the activity of ASK1 and preventing it from oligomerizing[21] .

 

Although most of the soluble cytoplasmic glutathione transferase isozymes are found in the cytoplasm, some are also located in mitochondria, the cell membrane or the nucleus. For example, mitochondrial glutathione transferase A4 has been reported in humans and mice. Studies have shown that mitochondrial glutathione transferase A4 is highly phosphorylated, translated and localized to mitochondria with the help of the molecular partner Hsp70; further studies have shown that this mitochondrial localization signal is located in the C-terminal region of the 20-position amino acid region and requires activation of the carbon-terminal phosphorylation site of protein kinase A (Ser-189) or protein kinase C (Thr-193) [22-23]. ] . Similarly, a highly homologous isoform of human glutathione transferase A1, called M-glutathione transferase A, was found in human liver microsomes, which is closely related to antioxidant damage of cell membranes[24] . The expression of mouse glutathione O1[25] and glutathione T2[26] in the nucleus has also been reported.

 

Unlike the tissue-specific expression of soluble glutathione transferase, the Kappa-like isozymes are widely expressed in various tissues, including liver, kidney, stomach, and heart, and have been shown to be associated with mitochondria of liver and kidney[27] , suggesting that mitochondrial glutathione transferase is the basis of cellular metabolism[28] . Glutathione transferase K1 is not only found in mitochondria, but also in peroxisomes. Given the importance of these two organelles in lipid metabolism, it is suggested that glutathione transferase K1 may be involved in the oxidative activation/transfer of fatty acid β as well as in the detoxification of lipid peroxides[6] . In addition, Morel et al. found that the C-terminal Ala-Arg-Leu sequence of glutathione transferase K1 is a signal for peroxisome targeting, suggesting that the C-terminal end has an important role in peroxisome targeting[6] . Although it has been suggested that glutathione transferase K1 may enter mitochondria with the help of a molecular chaperone, heat shock protein (Hsp60)[11] , or through the cleavage site at the N-terminal end of the mitochondrial conductor peptide[6] , the specific process of glutathione transferase K1 localization to the mitochondria needs to be further investigated.

 

Mitochondrial glutathione transferase is a complex family with diverse functions, including not only GSH-dependent transferase functions but also a series of hydrophobic compounds synthesis and transport. For example, M glutathione transferase 1 is mainly responsible for GSH-related transferase and isozyme-catalyzed reactions, similar to soluble glutathione transferase. It was found that M glutathione transferase 1 not only plays an important role in catalyzing the coupling of GSH with halogenated arenes and polyhalogenated unsaturated hydrocarbons[29] , but also participates in the metabolism of lipid hydroperoxides. It also participates in the metabolism of lipid hydroperoxides (e.g., fatty acid peroxides, phospholipid peroxides, etc.) [30-31], suggesting that M glutathione transferase 1 is an essential detoxifying enzyme in organisms, especially for the detoxification of exogenous toxic compounds and oxidative stress products. In addition, leukotriene C4 synthase (LTC4S), 5-lipoxygenase-activating protein (FLAP) and prostaglandin E2 synthase (PGES1) are involved in eicosanoids, eicosanoids and oxidative stress, respectively. They are involved in the synthesis of eicosanoids, leukotrienes and prostaglandins, respectively, and play a role in the arachidonoid pathway, whereas the M glutathione transferase 2 and M glutathione transferase 3 genes are responsible for the lowering of the (S)-5-peroxo-8,11,14-6-trans-didecatetraenoic acid[32] .

 

5 Glutathione transferase polymorphisms and disease

In recent years, extensive research on glutathione transferase gene polymorphisms and disease has contributed to the understanding of their important roles in carcinogen metabolism, antimutagenicity, antitumor, and apoptosis regulation, and has been important for the development of new disease defenses and overall therapeutic improvements. As early as 1988, Seidegård et al. identified three alleles of glutathione transferase M1, namely glutathione transferase M1 ∗0, glutathione transferase M1 ∗A, glutathione transferase M1 ∗B, in human liver tissues by conventional molecular cloning. Glutathione transferase M1 is mainly expressed in the liver, while the other members of the enzyme are expressed outside the liver. Further analysis revealed that 40% to 60% of the population had a homozygous deletion of the glutathione M1 ∗0 allele[33] , which puts this population at a higher risk of developing lung and colon cancer[34-35] .

 

Subsequently, two alleles of glutathione M3 were identified: glutathione M3 ∗A and glutathione M3 ∗B[36] , and two intronic SNPs were reported in glutathione M4[37] . Glutathione transferase P is highly expressed mainly in extrahepatic tissues and tumor cells[38] , with two SNP sites (I105V and A114V) and four alleles in the population, namely: glutathione transferase P ∗A (I105/A114), glutathione transferase P ∗B (V105/A114), glutathione transferase P ∗C (V105/V114) and glutathione transferase M3 ∗B[39] . ) and glutathione transferase P ∗D (I105/V114) [39-40] . The Val-105 variant increases the catalytic activity for aromatic epoxides [41] and is associated with a higher risk of testicular and bladder cancer [42]. Two glutathione transferase T alleles have been reported in the population: glutathione transferase T1 ∗ 0 and glutathione transferase T1 ∗ 1[43] , with the former completely lost in the population. In addition, 20% of Caucasians have a loss of allelic purity[44] , which makes them more susceptible to colon cancer, astrocytoma and myelodysplastic syndromes[45-47] . Glutathione transferase Z has three alleles in the population and contains two SNP sites: glutathione transferase Z1 ∗ A (A94A124), glutathione transferase Z1 ∗ B (A94 ~ G124), and glutathione transferase Z1 ∗ C (G94 ~ G124), and the different mutant proteins have different substrate binding properties [48]. M glutathione transferase 1 has four polymorphic sites, two of which are located in introns, one in the 3′ non-coding region, and the other in the promoter region; and people with the GG/GG (102G>A/16416G>A) genotype have a higher risk of developing colon cancer[49] .

 

6 Animal genetic engineering of glutathione transferase

Studies have shown that mouse glutathione transferase A4 has a strong catalytic activity towards 4-hydroxynonene (4-HNE). 4-HNE is a strong electrophile, a product of lipid peroxidation at the Michaelis receptor, and forms covalent adducts with proteins, nucleic acids and phospholipids. Engle et al. reported that mice with a pure mutation in glutathione transferase A4 were more susceptible to bacterial infections and increased susceptibility to paraquat (a herbicide), and the knockout mice showed significantly reduced conjugation of GSH with 4-hydroxynonenal (4-HNE) in tissues such as the brain and heart, suggesting that knockout of the glutathione transferase A4 gene reduces the detoxification function of the mice[50] . The knockout of glutathione transferase A4 decreased the detoxification function in mice[50] . However, the up-regulation of antioxidant response element (ARE)-related genes may be another compensatory mechanism for the knockdown of glutathione A4[51] . Further bioinformatics analyses showed that the 5′ upstream of glutathione A4 gene had an ARE similar to that of mouse quinone oxidoreductase 1 (NADPH) gene, which promoted 4-HNE metabolism and increased the level of glutathione A4 in the body, suggesting an important role of the mouse glutathione A4 gene in the fight against lipid peroxidation[52-53] .

 

The glutathione transferase M5 gene mainly encodes a brain/testis transferase, but it is not known what effect knockout has on the phenotype of mice[54] .

 

Pi-glutathione transferase has two coding genes in mice, glutathione transferase P1 and glutathione transferase P2, which play a major role in the detoxification of carcinogens, especially polycyclic aromatic hydrocarbons (PAH). The livers of mice mutated in glutathione P1 and glutathione P2 have lost transferase activity toward diuretic acid. In another study, the number of papillomas induced by dihydroxymethylbutyric acid and p-benzodicarboxylic acid in glutathione P1/P2-/- mice was nearly three times higher than that in normal mice, suggesting that glutathione transferase P plays an important role in resistance to exogenous compounds in carcinogenesis[55] . Surprisingly, glutathione transferase P1/P2-/- mice were more resistant to hepatotoxicity induced by the analgesic acetaminophen, which may be related to the faster GSH regeneration in the livers of double-knockout mice [56]. In addition, given the role of π-glutathione transferase in promoting the redox cycling of the acetaminophen metabolite, benzoquinone imine (NAPQI), it is hypothesized that deletion of π-glutathione transferase impairs the redox cycling of NAPQI, resulting in a decrease in GSH[56] .

 

Sigma-glutathione transferase encodes hematopoietic or GSH-dependent prostaglandin D2 synthetase (HPGDS), which is responsible for catalyzing the synthesis of an arachidonate-like prostaglandin D2 that plays an important role in immune responses; the knockout mice exhibit relatively weak allergic responses compared to wild-type mice[57] .

 

Glutathione transferase Z1 gene encodes maleylacetoacetate isomerase (MAAI), which is involved in the isomerization of MAAI in the phenylalanine/tyrosine metabolic pathway and is the penultimate catalytic step in tyrosine metabolism. Biochemical studies have shown that glutathione transferase Z1-/- mice have a reduced enzymatic response and impaired recognition of the substrates maleylacetone and chlorofluoroacetic acid[58] . Pathophysiological studies further revealed that glutathione transferase Z1-/- mice have reduced ability to metabolize both succinylacetone and other maleoylacetate-derived metabolites[59] . Interestingly, although glutathione transferase Z1 isoform-deficient mice (3% propylanilic acid-fed BALB/c mice) cause a range of pathologies, such as hepatic necrosis, steatosis, and peripheral leukemia, they also cause an increase in the expression of alpha-, mu-, and pi-class glutathione transferase as well as quinone oxidoreductase (NQO1), which may be the result of accumulation of tyrosine-lowering products. The increase in the expression of these enzymes may be due to the accumulation of tyrosine lowering products. It is noteworthy that all of these genes with elevated expression have ARE (antioxidant response element) or EpRE (electrophilic response element), and therefore, it is hypothesized that zeta-glutathione transferase also possesses antioxidant and electrophilic defenses. Overall, the dysfunction of tyrosine metabolism is the main cause of the reduced hepatic detoxification and antioxidant capacity of glutathione transferase Z1-/- mice[59] .

 

Mitochondrial glutathione transferase also has multiple functions in mammals, e.g., M glutathione transferase 1, M glutathione transferase 2, and M glutathione transferase 3 have detoxification functions, and FLAP, LTC4S, and PGES1 each play a role in the synthesis of lipoxygenase, leukotriene C4, and prostaglandin E2; furthermore, M glutathione transferase 2 and M glutathione transferase 3 also have a function in synthesizing leukotriene C4[60] . In addition, M glutathione transferase 2 and M glutathione transferase 3 also function in the synthesis of leukotriene C4[60] . Currently, studies of class I and IV microsomal glutathione transferase in genetically engineered mice have been reported, suggesting an important role for the MAPEG gene in allergic and inflammatory responses, but its function in oxidative stress has not yet been reported. In peritonitis induced by yeast glycan A, leukotriene C4 was not synthesized in the peritoneal lavage fluid of mutant mice but was significantly increased in wild-type mice, suggesting that leukotriene synthesis is lost in FLAP knockout mice. Importantly, metabolites of the 5-aliphatic oxygenase pathway, such as 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene A4, were not detected in FLAP knockout mice [61].

 

Since 5-lipoxygenase converts arachidonic acid to leukotriene A4 and further converts it to 5-HETE or binds it to GSH to produce cysteinyl leukotrienes, the above results suggest that FLAP plays a crucial role in the whole leukotriene synthesis process[61] . Knockdown of LTC4S gene reduces the binding capacity of LTA4 and GSH, which also affects leukotriene synthesis[62] . Macrophages in class IV PTGES knockout mice lost prostaglandin E2 synthesis compared to wild-type mice; PTGES mutant mice were found to have some defenses against fibroproliferation, inflammation, proteoglycan damage, cellular infiltration, and cartilage damage associated with collagen arthritis induced by type II collagen in chickens[63] . The study of neuronal febrile response in PTGES knockout mice showed that although the mice had perfect febrile ability, they did not show febrile response after infection with bacterial lipopolysaccharide and did not synthesize prostaglandin E2, suggesting that PTGES is the central switch of immune-induced fever and is expected to be a target for the treatment of fever[64] .

 

7 Conclusion

Glutathione transferase is a superfamily of proteases encoded by several genes that have multiple functions in the body's biotransformation, immunity and other defense systems, such as detoxification and antioxidant. Glutathione transferase s are found in a wide range of tissues and cells in various organisms and have been implicated in a variety of disease processes including cellular damage, hypoxia, toxicity, and aging. However, due to the existence of various isoforms, tissue-specific and substrate-specific expression, and interactions with other antioxidant enzymes, the research on glutathione transferases has been slow. With the continuous development and application of molecular biology and bioinformatics, the sequence, evolutionary diversity, molecular structure, and antioxidant mechanism of mammalian glutathione transferase s can be better elucidated and implemented.

 

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