The human hGSTA5 gene encodes an enzymatically active protein.

BACKGROUND: Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. METHODS: Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. RESULTS: Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. CONCLUSIONS: hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. GENERAL SIGNIFICANCE: Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.

Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2.

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.

Disruption of the mGsta4 gene increases life span of C57BL mice.

The lipid peroxidation product 4-hydroxynonenal (4-HNE) forms as a consequence of oxidative stress. By electrophilic attack on biological macromolecules, 4-HNE mediates signaling or may cause toxicity. A major route of 4-HNE disposal is via glutathione conjugation, in the mouse catalyzed primarily by glutathione transferase mGSTA4-4. Unexpectedly, mGsta4-null mice, in which 4-HNE detoxification is impaired, have an extended life span. This finding could be explained by the observed activation of the transcription factor Nrf2 in the knockout mice, which in turn leads to an induction of antioxidant and antielectrophilic defenses. Especially, the latter could provide a detoxification mechanism that contributes to enhanced longevity. We propose that disruption of 4-HNE conjugation elicits a hormetic response in which an initially increased supply of 4-HNE is translated into activation of Nrf2, leading to a new steady state in which the rise of 4-HNE concentrations is dampened, but life-extending detoxification mechanisms are concomitantly induced.

Fat accumulation in Caenorhabditis elegans triggered by the electrophilic lipid peroxidation product 4-hydroxynonenal (4-HNE).

Deposition and mobilization of fat in an organism are tightly controlled by multiple levels of endocrine and neuroendocrine regulation. Because these hormonal mechanisms ultimately act by affecting biochemical reactions of fat synthesis or utilization, obesity could be also modulated by altering directly the underlying lipid biochemistry. We have previously shown that genetically modified mice with an elevated level of the lipid peroxidation product 4-HNE become obese. We now demonstrate that the process is phylogenetically conserved and thus likely to be universal. In the nematode C. elegans, disruption of either conjugation or oxidation of 4-HNE leads to fat accumulation, whereas augmentation of 4-HNE conjugation results in a lean phenotype. Moreover, direct treatment of C. elegans with synthetic 4-HNE causes increased lipid storage, directly demonstrating a causative role of 4-HNE. The postulated mechanism, which involves modulation of acetyl-CoA carboxylase activity, could contribute to the triggering and maintenance of the obese phenotype on a purely metabolic level.

Role of the electrophilic lipid peroxidation product 4-hydroxynonenal in the development and maintenance of obesity in mice.

The lipid peroxidation product 4-hydroxynonenal (4-HNE) is a signaling mediator with wide-ranging biological effects. In this paper, we report that disruption of mGsta4, a gene encoding the 4-HNE-conjugating enzyme mGSTA4-4, causes increased 4-HNE tissue levels and is accompanied by age-dependent development of obesity which precedes the onset of insulin resistance in 129/sv mice. In contrast, mGsta4 null animals in the C57BL/6 genetic background have normal 4-HNE levels and remain lean, indicating a role of 4-HNE in triggering or maintaining obesity. In mGsta4 null 129/sv mice, the expression of the acetyl-CoA carboxylase (ACC) transcript is enhanced several-fold with a concomitant increase in the tissue level of malonyl-CoA. Also, mitochondrial aconitase is partially inhibited, and tissue citrate levels are increased. Accumulation of citrate could lead to allosteric activation of ACC, further augmenting malonyl-CoA levels. Aconitase may be inhibited by 4-HNE or by peroxynitrite generated by macrophages which are enriched in white adipose tissue of middle-aged mGsta4 null 129/sv mice and, upon lipopolysaccharide stimulation, produce more reactive oxygen species and nitric oxide than macrophages from wild-type mice. Excessive malonyl-CoA synthesized by the more abundant and/or allosterically activated ACC in mGsta4 null mice leads to fat accumulation by the well-known mechanisms of promoting fatty acid synthesis and inhibiting fatty acid beta-oxidation. Our findings complement the recent report that obesity causes both a loss of mGSTA4-4 and an increase in the level of 4-HNE [Grimsrud, P. A., et al. (2007) Mol. Cell. Proteomics 6, 624-637]. The two reciprocal processes are likely to establish a positive feedback loop that would promote and perpetuate the obese state.

Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme.

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1-mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67-78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic.

We have shown previously that the chemoprotective activity of diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, against benzo[a]pyrene (BP)-induced forestomach carcinogenesis in mice correlates strongly with its inductive effects on the expression of Pi class glutathione (GSH) transferase mGSTP1-1. The present structure-activity relationship studies were designed to define the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS. Hepatic mGSTP1 mRNA levels rose rapidly upon treatment of mice with DADS, reached a maximum between 12 and 24 h (< or =5.7-fold induction) and fell to control levels by 48 h after DADS treatment. Induction of mGSTP1 mRNA in the forestomach was maximal between 6 and 12 h after DADS treatment (< or =4.7-fold induction). The mGSTP1 mRNA expression was either unaltered (liver) or moderately increased (forestomach) upon treatment of mice with dipropyl disulfide (DPDS), which is a naturally occurring saturated analog of DADS. These results indicated that the allyl groups are critical for the mGSTP1-inducing activity of DADS. A statistically significant increase in the expression of mGSTP1 mRNA was also observed in the liver and forestomach of mice treated with diallyl monosulfide (DAMS), albeit to a much lesser extent compared with DADS. These results indicated that the oligosulfide chain length in garlic organosulfides (OSCs) is equally important for their mGSTP1-inducing activity. The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further investigated by testing a pair of alkadienes (1,7-octadiene and 1,8-nonadiene) having structural similarity to DADS. Both DADS and the alkadienes carry allyl groups at both ends of a linear molecule and the distance between the allylic carbon atoms is similar in both compounds, but the central disulfide chain of DADS is replaced with an alkyl chain in the alkadienes. The alkadienes were either ineffective or moderately active in increasing mGSTP1 expression. In conclusion, the results of the present study clearly indicate that the presence of terminal allyl groups as well as the central disulfide chain is required for maximum induction of mGSTP1 in vivo by garlic-derived OSCs.