Glutathione S-Transferase pi Mediates MPTP-Induced c-Jun N-Terminal Kinase Activation in the Nigrostriatal Pathway

Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.     

谷胱甘肽硫转移酶基因表达的调控

催化内源性或外源性亲电子化合物与谷胱甘肽(GSH)结合的谷胱甘肽硫转移酶(GST)超基因家族是一族解毒功能蛋白.其基因的表达通过不同的机制受多种物质的调控.根据最近文献资料,对调控谷胱甘肽硫转移酶基因表达的基因结构、调控机制及氧化应激对谷胱甘肽硫转移酶基因表达的调控作用等作一简要综述.     

谷胱甘肽S-转移酶的诱导表达及提纯

含有日本血吸虫谷胱甘肽S -转移酶基因的质粒 pGEX - 5X - 1在大肠杆菌BL2 1 (DE3)菌株中得到表达。 37℃[1 ]下用 1 %乳糖诱导谷胱甘肽S -转移酶 (GST)的最适表达时间为 3h。盐析粗提酶液并以Habig法[2 ] 监测GST的活性 ,用pH6 .0 ,饱和度为 30 %硫酸铵去除杂蛋白 ,并调 pH7.5 ,饱和度 70 %硫酸铵使GST大量析出并保持活性。用透析法脱盐 ,并用Sephadex -G5 0凝胶对GST进行了初步纯化。初步探讨了GST的保存方法     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

谷胱甘肽S－转移酶基因P1的研究进展

谷胱甘肽S－转移酶P1－1在癌变细胞和抗药性肿瘤细胞中表达水平发生变化,提示可以作为恶性转化及肿瘤抗药性的标志物．对大鼠谷胱甘肽S－转移酶P1基因上游调控序列的研究发现在－2.5kb及－2.2kb各存在一增强子序列GPEⅠ,GPEⅡ,－400bP存在一沉寂子．GPEⅠ、沉寂子上均至少结合有3种反式作用因子．人谷胱甘肽S－转移酶P1基因上游区域中迄今尚未发现增强子或沉寂子,但却发现了胰岛素及视黄酸的应答序列,在癌变细胞和抗药性的肿瘤细胞中该基因表达的调控机制有别于正常细胞．     

人胎盘谷胱甘肽S-转移酶动力学及抑制剂的研究

研究了人胎盘型谷胱甘肽S-转移酶(GST-π)的动力学。底物GSH和1-氯-2,4-二硝基苯(CDNB)的km分别为0.109和0.870mmol/L。苯唑青霉素和先锋霉素Ⅰ能抑制GST—π,以先锋霉素较明显,属非竞争性抑制。溴磺酜对CDNB也是非竞争作用,但胆红素则对CDNB竞争而对GSH非竞争地抑制酶活力。S-正辛烷和S-正已烷谷胱甘肽与GSH竞争而与CDNB非竞争地抑制GST-π。已充分证明GST-π所催化的双底物反应属随机顺序机制。化学修饰实验发现:巯基、胍基、氨基、羧基和吲哚基可能参与酶活性中心的组成。     

The Effects of Methylglyoxal on Glutathione S-Transferase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes that accumulate in plants under environmental stress. Glutathione S-transferases (GSTs) play important roles, including detoxification, in the stress tolerance systems of plants. To determine the effects of MG, we characterized recombinant GST. MG decreased GST activity and thiol contents with increasing K _m. GST can serve as a target of MG modification, which is suppressed by application of reduced glutathione.     

Extensive Functional Diversification of the Populus Glutathione S-Transferase Supergene Family

Identifying how genes and their functions evolve after duplication is central to understanding gene family radiation. In this study, we systematically examined the functional diversification of the glutathione S-transferase (GST) gene family in Populus trichocarpa by integrating phylogeny, expression, substrate specificity, and enzyme kinetic data. GSTs are ubiquitous proteins in plants that play important roles in stress tolerance and detoxification metabolism. Genome annotation identified 81 GST genes in Populus that were divided into eight classes with distinct divergence in their evolutionary rate, gene structure, expression responses to abiotic stressors, and enzymatic properties of encoded proteins. In addition, when all the functional parameters were examined, clear divergence was observed within tandem clusters and between paralogous gene pairs, suggesting that subfunctionalization has taken place among duplicate genes. The two domains of GST proteins appear to have evolved under differential selective pressures. The C-terminal domain seems to have been subject to more relaxed functional constraints or divergent directional selection, which may have allowed rapid changes in substrate specificity, affinity, and activity, while maintaining the primary function of the enzyme. Our findings shed light on mechanisms that facilitate the retention of duplicate genes, which can result in a large gene family with a broad substrate spectrum and a wide range of reactivity toward different substrates.     

谷胱甘肽S-转移酶Zeta类基因在酿酒酵母中的表达

谷胱甘肽S-转移酶Zeta类基因在酿酒酵母中的表达 贾向东1,陈喜文1,陈德富1,陈洁2 (1.南开大学生命科学学院,生物活性材料教育部重点实验室,天津300071;2.湖南怀化市铁路第一中学,怀化418000) 摘要：谷胱甘肽S-转移酶Zeta类(GSTZ)是一种重要的多功能酶,与细胞生化代谢、环境净化等密切相关。将拟南芥、甘蓝型油菜品系陕2B与垦C1的GSTZ基因克隆到大肠杆菌—酿酒酵母穿梭表达载体pYES2的多克隆位点,筛选到重组子后,提取重组质粒并将其转入酿酒酵母营养缺陷型菌株INCSc1细胞中,经SC-U培养基选择得到重组酵母Y2At、Y2BnB和Y2BnC。重组酵母在含棉子糖和半乳糖的诱导培养基中,表达出了具有二氯乙酸脱氯活力的谷胱甘肽S-转移酶Zeta类,且主要以可溶状态存在于酵母细胞中。不同碳源比较发现,使用半乳糖为唯一碳源时,与棉子糖和半乳糖共同使用相比,酵母生长虽受到轻微影响,但表达的GSTZ比活力几乎不受任何影响。0~96h诱导时间的优化实验表明,36h诱导下呈现最高比活力。同时也对不同GSTZ的Km值进行了比较。     

Evaluation of Glutathione S-Transferase GSTM1 and GSTT1 Deletion Polymorphisms on Type-2 Diabetes Mellitus Risk

Background

Due to the activity of GSTs in the detoxification of oxidative stress products, deletion polymorphisms of GSTM1 and GSTT1 may contribute to susceptibility to T2DM, since B-cells express very low levels of antioxidant enzymes. Recently, some studies have shown an association between GSTM1-null/GSTT1-null genotypes and an increased susceptibility to T2DM. A relationship between these polymorphisms and changes in the clinical parameters of diabetic patients has also been investigated. However, the results diverge considerably among the studies. Thus, this case-control study was designed to contribute to existing knowledge, as there are no studies on this issue performed in the Brazilian population.

Methods and Findings

A total of 120 patients and 147 healthy individuals were included in this study. GSTT1 and GSTM1 deletion polymorphisms were genotyped by multiplex SYBR Green Real-Time PCR. The GSTT1-null genotype conferred a 3.2-fold increased risk to T2DM relative to the present genotype. There was no association between GSTM1-null and T2DM risk. In diabetic patients, GSTT1-null conferred higher levels of triglycerides and VLDL-cholesterol, while GSTM1-null was associated with increased levels of fasting blood glucose, glycated hemoglobin and blood pressure. We emphasized a necessity for applying log-linear analysis in order to explore an interaction between these polymorphisms properly.

Conclusion

These results suggest that the GSTT1 polymorphism may play an important role in the pathogenesis of T2DM in the Brazilian population. This gene could then be added to a set of genetic markers to identify individuals with an increased risk for developing T2DM and complications associated with dyslipidemia in diabetic patients. Although there was no association of GSTM1 deletion polymorphism with susceptibility to T2DM, the influence of this polymorphism on important clinical parameters related to glycemia and blood pressure levels was verified. This finding suggests that both GSTM1-null and GSTT1-null may contribute to the clinical course of T2DM patients.     

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Glutathione S-transferases (GSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max L.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic GST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic GST but did not affect the intracellular isoforms. The discovery of apoplastic inducible GST will be discussed in light of the putative function of these enzymes in plants.     

过量表达谷胱甘肽转移酶基因对转基因拟南芥抗旱能力的影响

盐地碱蓬谷胱甘肽转移酶基因（OST）在拟南芥中过量表达后,在干旱胁迫下,转基因拟南芥植株的干重比野生型植株高,其总谷胱甘肽含量和谷胱甘肽库的氧化水平都比野生型植株的高,而丙二醛含量则比野生型的低。这些显示转基因拟南芥的抗干旱胁迫能力有所增强。     

Optimization of Storage and Stability of Camel Liver Glutathione S-Transferase

Glutathione S-transferases (GSTs) are multifunctional enzymes and play an important role in cellular detoxification. Besides this, GSTs act as cytosolic carrier proteins that bind hydrophobic compounds such as heme, bilirubin, steroids, and polycyclic hydrocarbons. GST has great importance in biotechnology, as it is a target for vaccine and drug development and biosensors development for xenobiotics. Moreover, the GST tag has been extensively used for protein expression and purification. Until now, biophysical properties of camel liver GST have not been characterized. In the present study we have purified camel (Camelus dromedarius) liver GST to homogeneity in a single step by affinity chromatography with 23.4-fold purification and 60.6% yield. Our results showed that maximal activity of GST was at pH 6.5 and it was stable in the pH range of 5 to 10. The optimum temperature was 55°C and the T_m was 57°C. The chemical chaperone glycerol (3.3 M) was able to protect GST activity and aggregation against thermal denaturation by stabilizing the protein structure at 50 and 57°C, respectively. However, L-arginine (125 mM) did not protect GST against thermal stress. Far-ultraviolet circular dichroism (CD) spectra showed that glycerol protected the secondary structure of GST while L-arginine induced conformational changes under thermal stress. In conclusion, our studies on the GST stability suggest that glycerol works as a stabilizer and L-arginine acts as a destabilizer.     

Xenobiotic Binding Domain of Glutathione S-Transferase Has Cryptic Antimicrobial Peptides

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides are one of the important components of innate immune defense system and play a critical role in controlling...     

Glutathione S-Transferase P Influences Redox and Migration Pathways in Bone Marrow

To interrogate why redox homeostasis and glutathione S-transferase P (GSTP) are important in regulating bone marrow cell proliferation and migration, we isolated crude bone marrow, lineage negative and bone marrow derived-dendritic cells (BMDDCs) from both wild type (WT) and knockout (Gstp1/p2^−/−) mice. Comparison of the two strains showed distinct thiol expression patterns. WT had higher baseline and reactive oxygen species-induced levels of S-glutathionylated proteins, some of which (sarco-endoplasmic reticulum Ca²⁺-ATPase) regulate Ca²⁺ fluxes and subsequently influence proliferation and migration. Redox status is also a crucial determinant in the regulation of the chemokine system. CXCL12 chemotactic response was stronger in WT cells, with commensurate alterations in plasma membrane polarization/permeability and intracellular calcium fluxes; activities of the downstream kinases, ERK and Akt were also higher in WT. In addition, expression levels of the chemokine receptor CXCR4 and its associated phosphatase, SHP-2, were higher in WT. Inhibition of CXCR4 or SHP2 decreased the extent of CXCL12-induced migration in WT BMDDCs. The differential surface densities of CXCR4, SHP-2 and inositol trisphosphate receptor in WT and Gstp1/p2^−/− cells correlated with the differential CXCR4 functional activities, as measured by the extent of chemokine-induced directional migration and differences in intracellular signaling. These observed differences contribute to our understanding of how genetic ablation of GSTP causes higher levels of myeloproliferation and migration.     

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.