Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2)

Tan, K. Hong; Board, Philip G.

doi:10.1042/bj3150727

Cited by 66 publications

(50 citation statements)

References 29 publications

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“…Unfortunately attempts in multiple laboratories to demonstrate glutathione S-transferase enzyme activity for Ure2 have been unsuccessful (7,16,20). Although negative results are rarely reported in detail, the lack of success might derive from inherent instability in the enzyme, an observed characteristic of glutathione S-transferases, or from performing the assay with substrates that are not the preferred ones for the putative transferase (25)(26)(27)(28). The difficulties experienced in attempts to assay an enzyme activity prompted us to take a step backward from in vitro assays and ask more simply whether Ure2 is required for protection of cells against the growth-inhibitory effects of compounds reported to be glutathione S-transferase substrates in other organisms.…”

Section: Ure2 Is Required For Protection Against Heavy Metals-ure2mentioning

confidence: 99%

“…Two compounds in this category are hydrogen peroxide and diamide. Hydrogen peroxide is detoxified in two ways: by peroxidation, an activity found in some mammalian Theta-class GSTs and in S. pombe GST3 or by GSH conjugation of cellular products that are oxidized by hydrogen or other peroxides (25,26,28). Diamide also oxidizes cellular proteins and other constituents but, in addition, depletes the reduced glutathione pool because it is detoxified via glutathione-dependent reduction (36).…”

Section: Ure2 Detoxifies Heavy Metals In S Cerevisiaementioning

confidence: 99%

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Ure2, a Prion Precursor with Homology to Glutathione S-Transferase, Protects Saccharomyces cerevisiae Cells from Heavy Metal Ion and Oxidant Toxicity

Rai

Tate

Cooper

2003

Journal of Biological Chemistry

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Ure2, the protein that negatively regulates GATA factor (Gln3, Gat1)-mediated transcription in Saccharomyces cerevisiae, possesses prion-like characteristics. Identification of metabolic and environmental factors that influence prion formation as well as any activities that prions or prion precursors may possess are important to understanding them and developing treatment strategies for the diseases in which they participate. Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases. However, multiple attempts over nearly 2 decades to demonstrate Ure2-mediated S-transferase activity have been unsuccessful, leading to the possibility that Ure2 may well not participate in glutathionation reactions. Here we show that Ure2 is required for detoxification of glutathione S-transferase substrates and cellular oxidants. ure2⌬ mutants are hypersensitive to cadmium and nickel ions and hydrogen peroxide. They are only slightly hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to 1-chloro-2,4-dinitrobenzene, the most commonly used substrate for glutathione S-transferase enzyme assays. Therefore, Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations possess the same phenotypes as mutations in known S. cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes. These findings are consistent with Ure2 serving as a glutathione S-transferase in S. cerevisiae.Several neurodegenerative conditions derive from the same pathogenetic mechanism, i.e. a change in protein conformation, polymerization, and plaque formation. These conditions have been called conformational diseases such as Alzheimer's and the prionoses. Recent studies have demonstrated that a protein associated with such disease, amyloid ␤-protein, protects neurons from metal-induced oxidative damage (1). Neither the molecular basis for this activity nor how it is affected by environment and neuronal metabolism is yet known. The use of eukaryotic model systems, such as the yeast Saccharomyces cerevisiae, has greatly facilitated our acquisition of information about mammalian proteins, their functions and interactions, and how their synthesis and activities are regulated and integrated. In particular, the genetic study of prions has been

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Section: Ure2 Is Required For Protection Against Heavy Metals-ure2mentioning

confidence: 99%

Section: Ure2 Detoxifies Heavy Metals In S Cerevisiaementioning

confidence: 99%

Ure2, a Prion Precursor with Homology to Glutathione S-Transferase, Protects Saccharomyces cerevisiae Cells from Heavy Metal Ion and Oxidant Toxicity

Rai

Tate

Cooper

2003

Journal of Biological Chemistry

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“…2 Briefly, the plasmid contains a gene encoding a ubiquitin-3D pol fusion protein under control of the bacteriophage T7 RNA polymerase promoter (20). Escherichia coli, BL21(DE3), co-transformed with the 3D pol expression plasmid and plasmid pCG1, which directs the expression of a ubiquitin protease (20), were grown to A 600 ϭ 0.8 in NZCYM medium (Life Technologies, Inc.), and expression was induced by addition of isopropyl-1-thio-␤-D-galactopyranoside to 500 M. Cells were harvested by centrifugation after 4 h. 3D…”

Section: Materials Expression and Purification Of 3d Polmentioning

confidence: 99%

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

Arnold

Cameron

1999

Journal of Biological Chemistry

109

105

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We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D pol . Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D pol -catalyzed nucleotide incorporation and RNA recombination are discussed.Positive-strand RNA viruses cause a variety of diseases in humans ranging from the common cold (1) to chronic hepatitis (2). Critical to the replication of the genomes of these viruses is the virus-encoded, RNA-dependent RNA polymerase (RdRP) 1 (3). As this enzymatic activity is unique to virus-infected cells, the viral RdRP represents a very attractive target for the design of antiviral agents to treat RNA virus infection. Most positive-strand RNA viruses are thought to have the same general replication strategy, and this belief is generally extended to include the mechanism of genome replication (3). RdRPs from many viruses have been characterized to some extent (3-9). However, in no instance is detailed, mechanistic information available. This fact is even true for viruses, such as poliovirus, for which genetic and biochemical systems to study genome replication have existed for many years (10).Notwithstanding, poliovirus is one of the best understood systems with respect to the biochemistry of genome replication and is, therefore, an invaluable paradigm for all positivestrand RNA viruses (10). Replication of poliovirus genome initiates within the poly(A)-tract at the 3Ј-end of genomic RNA from a complex comprising viral factors (3AB, 3CD, 3B, and 3D pol ) (11-13) and possibly cellular factors (14). 3AB and 3CD are required primarily to establish an initiation complex, possibly by recruiting the polymerase, 3D pol (13). 3AB is a RNAbinding protein capable of interacting with 3D pol (15,16) that possibly stimulates the rate of elongation of nascent RNA (17) or enhances the efficiency of primer utilization (18)....

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“…Importantly, not only did SCR23 reconstitute the CMAC activity of rGSTT2-2, it also led to a new specificity for ethacrynic acid that is absent in both parents. This activity is equivalent to that exhibited by hGSTT2-2, the only other known theta-class GST capable of catalyzing this reaction (30). The evolution of this activity in the absence of an associated selection pressure suggests a potential role for low-homology recombination in the development of promiscuous protein functions, a topic that has been explored in detail for random mutagenesis and conventional family shuffling (31).…”

Section: Discussionmentioning

confidence: 97%

Evolution of highly active enzymes by homology-independent recombination

Griswold

Kawarasaki

Ghoneim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The theta-class GST enzymes hGSTT1-1 (human GST -1-1) and rGSTT2-2 (rat GST -2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C͞Os) at positions not accessible by DNA family shuffling. Highthroughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C͞Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k cat with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and lowhomology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme ''humanization.'' enzyme engineering ͉ enzyme humanization ͉ GST ͉ ITCHY ͉ high-throughput screening G STs play a crucial role in cellular detoxification by conjugating reactive electrophilic compounds to the tripeptide glutathione (GSH) (1). Mammals encode at least seven distinct classes of GSTs. The theta enzymes represent the oldest class of GSTs and, as such, are produced by a surprisingly diverse array of animals, plants, algae, and bacteria (2). Although theta-class enzymes retain the highly conserved GST 3D fold, they possess a characteristic C-terminal ␣-helical extension, which, unlike that of some alpha-class enzymes, covers both the electrophile and GSH-binding sites, thus clearly differentiating the theta class from other members of the soluble GST superfamily (3).The rat GST -2-2 (rGSTT2-2, 244 aa) and human GST -1-1 (hGSTT1-1, 240 aa) enzymes exhibit 54.3% overall amino acid identity. The GSH-binding domain, or G site, of the two enzymes (residues Ϸ1-77) is conserved (79.2% amino acid identity), and the 5Ј regions that encode this domain (nucleotides 1-231) exhibit 74.5% DNA sequence identity. In contrast, the electrophilic substrate binding domain, or H site (amino acid Ϸ89 to termini), of the rat and human enzymes (encoded by nucleotides Ϸ265-732 in rGSTT2-2 and nucleotides Ϸ265-720 in hG-STT1-1) exhibit only 41.4% amino acid identity and 57.9% DNA sequence identity. The sequence divergence of the two enzymes in the C-terminal domain is manifest in their respective electrophilic substrate selectivities. The rat enzyme preferentially catalyzes the GSH conjugation of 1-menaphythyl sulfate, whereas hGSTT1-1 exhibits a characteristic reactivity with dichloromethane (2). Additionally, we recently showed that rG-STT2-2 e...

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Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2)

Cited by 66 publications

References 29 publications

Ure2, a Prion Precursor with Homology to Glutathione S-Transferase, Protects Saccharomyces cerevisiae Cells from Heavy Metal Ion and Oxidant Toxicity

Ure2, a Prion Precursor with Homology to Glutathione S-Transferase, Protects Saccharomyces cerevisiae Cells from Heavy Metal Ion and Oxidant Toxicity

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

Evolution of highly active enzymes by homology-independent recombination

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