Glutathione S-transferase π localizes in mitochondria and protects against oxidative stress

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST π, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST π is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST π and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST π completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST π. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST π diminished these changes. GST π-targeting siRNA abolished the protective effect of GST π on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST π under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST π protects mitochondria against oxidative stress.     

Dual localization of glutathione S-transferase in the cytosol and mitochondria: implications in oxidative stress, toxicity and disease

Glutathione (GSH) conjugating enzymes, glutathione S-transferases (GSTs), are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus and plasma membrane. The regulation and function of GSTs have implications in cell growth, oxidative stress as well as disease progression and prevention. Of the several mitochondria localized forms, GSTK (GST kappa) is mitochondria-specific since it contains N-terminal canonical and cleavable mitochondria targeting signals. Other forms like GST alpha, mu and pi purified from mitochondria are similar to the cytosolic molecular forms or 'echoproteins'. Altered GST expression has been implicated in hepatic, cardiac and neurological diseases. Mitochondria-specific GSTK has also been implicated in obesity, diabetes and related metabolic disorders. Studies have shown that silencing the GSTA4 (GST alpha) gene resulted in mitochondrial dysfunction, as was also seen in GSTA4 null mice, which could contribute to insulin resistance in type 2 diabetes. This review highlights the significance of the mitochondrial GST pool, particularly the mechanism and significance of dual targeting of GSTA4-4 under in vitro and in vivo conditions. GSTA4-4 is targeted in the mitochondria by activation of the internal cryptic signal present at the C-terminus of the protein by protein-kinase-dependent phosphorylation and cytosolic heat shock protein (Hsp70) chaperone. Mitochondrial GST pi, on the other hand, has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N-terminal region. Both physiological and pathophysiological implications of GST translocation to mitochondria are discussed in the review.     

Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.     

Alleviation of iron induced oxidative stress by the grape fruit flavanone naringin in vitro

Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. The protective effect of naringin, a grape fruit flavanone, was studied in iron overloaded isolated mouse liver mitochondria, where the isolated mitochondrial fraction was incubated with various concentrations of naringin before ferric ion loading. Iron overloading of mitochondrial fraction resulted in an increase in lipid peroxidation, protein oxidation, and DNA damage, whereas iron overload reduced the glutathione (GSH) concentration, glutathione-S-transferase (GST), glutathione peroxidase (GSHPx), catalase and superoxide dismutase (SOD) activities. Pretreatment of mitochondrial fraction with naringin inhibited iron-induced lipid peroxidation, protein oxidation, and DNA damage. Conversely, naringin supplementation arrested iron-induced depletion in the GSH contents, GSHPx, GST, SOD and catalase activities significantly. Ferric iron reduction assay revealed that naringin could not reduce ferric iron into ferrous iron indicating that it did not exhibit prooxidant activity. Iron free coordination site assay indicated that naringin was unable to occupy all the active sites of iron indicating that naringin did not completely chelate iron. Our study demonstrates that naringin was able to share the burden of endogenous oxidants by inhibiting the iron-induced depletion of all important antioxidant enzymes as well as GSH and may act as a good antioxidant.     

Oxidative stress in zinc-induced dopaminergic neurodegeneration: implications of superoxide dismutase and heme oxygenase-1

The study was undertaken to investigate the effect of zinc (Zn) on glutathione S-transferase (GST) and superoxide dismutases (SOD) activities and on the expressions of cytosolic Cu, Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), γ-glutamyl cysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) in the nigrostriatal tissue of rats. Additionally, Zn-induced alterations in the neurobehavioral parameters, lipid peroxidation (LPO), striatal dopamine and its metabolites and tyrosine hydroxylase (TH) protein expression were measured to assess their correlations with the oxidative stress. Zn exposure reduced the locomotor activity, rotarod performance, striatal dopamine and its metabolites and TH protein expression. LPO, total SOD, SOD1 and SOD2 activities were increased while GST and catalase were reduced in a dose and time dependent manner. Expressions of SOD1 and HO-1 were increased while no change was observed in SOD2 and γ-GCS expressions. The results obtained suggest that Zn-induced augmentation of total SOD, SOD1, SOD2 and HO-1 was associated with increased oxidative stress and neurodegenerative indexes indicating the involvement of both cytosolic and mitochondrial machinery in Zn-induced oxidative stress leading to dopaminergic neurodegeneration.     

Expression and purification of a novel rice (Oryza sativa L.) mitochondrial ATP synthase small subunit in Escherichia coli

To clarify the function of the rice mitochondrial ATP synthase 6 kDa subunit (RMtATP6), a method of producing large quantities of this protein is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of RMtATP6. The RMtATP6 gene (GenBank Accession No. ) was cloned into the pGEX-6p-3 vector to allow expression of RMtATP6 as a glutathione S-transferase (GST) fusion protein. The RMtATP6-GST fusion protein was purified by affinity chromatography using a glutathione-Sepharose 4B column. A Western blot analysis using anti-GST antibody showed that the fusion protein was not degraded. After enzymatic cleavage of the GST tail, the RMtATP6 protein showed a molecular weight of around 6 kDa. The predicted pI of this protein is 10.01. After improving the conditions of expression and the purification procedures, the final yield of the entire expression and purification process was about 4.6 mg of pure RMtATP6 protein per liter of bacterial culture.     

高温下线粒体小分子热激蛋白对柠檬酸合成酶、线粒体和花粉粒的保护作用

选用pGEX-6P-1表达载体,构建GST与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白.分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效.利用转基因方法,将线粒体小分子热激蛋白基因导入烟草,在CaMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用.此外,转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力.     

Oxidative stress in zinc-induced dopaminergic neurodegeneration: Implications of superoxide dismutase and heme oxygenase-1

Abstract

The study was undertaken to investigate the effect of zinc (Zn) on glutathione S-transferase (GST) and superoxide dismutases (SOD) activities and on the expressions of cytosolic Cu, Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), γ-glutamyl cysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) in the nigrostriatal tissue of rats. Additionally, Zn-induced alterations in the neurobehavioral parameters, lipid peroxidation (LPO), striatal dopamine and its metabolites and tyrosine hydroxylase (TH) protein expression were measured to assess their correlations with the oxidative stress. Zn exposure reduced the locomotor activity, rotarod performance, striatal dopamine and its metabolites and TH protein expression. LPO, total SOD, SOD1 and SOD2 activities were increased while GST and catalase were reduced in a dose and time dependent manner. Expressions of SOD1 and HO-1 were increased while no change was observed in SOD2 and γ-GCS expressions. The results obtained suggest that Zn-induced augmentation of total SOD, SOD1, SOD2 and HO-1 was associated with increased oxidative stress and neurodegenerative indexes indicating the involvement of both cytosolic and mitochondrial machinery in Zn-induced oxidative stress leading to dopaminergic neurodegeneration.     

Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.     

(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

A modified method for the purification of active large enzymes using the glutathione S-transferase expression system

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.     

Phosphorylation of Glutathione-S-transferase by Protein Kinase C-α Implications for Affinity-Tag Purification

Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2,500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein-protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-alpha (PKC-alpha) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.     

GST/ AEP 融合蛋白原核表达载体的构建、表达及鉴定

目的：为进一步研究抗癫痫肽（And—epilepsy peptide,AEP）的抗痫机制及筛选其相关作用蛋白,进行GST／AEP融合蛋白原核表达载体的构建及融合蛋白的表达。方法：通过PCR基因扩增对AEP基因进行扩增,并将其克隆于谷胱甘肽-S-转移酶（GST）融合蛋白表达质粒pGEX-4T-1中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌B121（DE3）,经IPTG诱导获得表达,并采用Western Blot进行检测。结果：成功构建了AEP原核表达载体,并在大肠杆菌B121中获得表达。结论：成功构建了GST／AEP原核表达载体,并表达了GST／AEP融合蛋白。     

Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.     

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex

The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein–protein and protein–RNA interactions by means of GST pull-down experiments. A total of 19 direct protein–protein and six direct protein–RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.     

游仆虫Rab蛋白基因的克隆表达与纯化

为对单细胞原生动物纤毛虫中Rab蛋白的功能进行研究 ,进而探讨以胞吞和胞吐为主要物质交换途径的纤毛虫中囊泡定向运输的机理 .利用PCR技术从游仆虫大核DNA及cDNA中扩增出rab基因 ,并进行了序列分析 ,该基因全长为 783bp ,两端为端粒序列 ,编码框为 6 2 4bp ,编码 2 0 7个氨基酸 ,开放读框中有 3个TGA ,在此编码半胱氨酸 .利用定点突变将rab基因中 3个TGA突变为通用半胱氨酸密码子TGC .将游仆虫Rab蛋白基因构建于原核表达载体pGEX 4T 2中 ,得到的重组质粒pGEX Eorab1转化至大肠杆菌BL2 1(DE3)中 ,IPTG诱导表达 .表达产物与抗GST抗体在 4 9kD处有很强的交叉反应 .融合蛋白GST EoRab1通过亲和层析柱纯化和凝血酶的切割 ,再经两步纯化得到电泳纯的游仆虫Rab蛋白 .     

A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate

Glutathione S-transferase (GST) is widely used to prepare and purify GSTtagged fusion proteins. Although GST improves protein solubility, detergents must often be used to achieve protein solubilization from bacterial lysates. However, purification of GST by affinity chromatography cannot be achieved in the presence of even low concentrations of the detergent sodium dodecyl sulfate (SDS). Here we show that 2-methyl-2,4-pentanediol (MPD) can prevent SDS from interfering with purification of GST, thus enabling purification of proteins that require SDS to improve their solubility.