Secretory type of recombinant thioredoxin h induces ER stress in endosperm cells of transgenic rice

Thioredoxin h (TRX h) functions as a reducing protein and is present in all organisms. As a new approach for inducing the endoplasmic reticulum (ER) stress, TRX h (OsTRX23) was expressed as a secretory protein using the endosperm-specific glutelin GluB-1 promoter and a signal peptide. In transgenic rice seeds, the majority of the recombinant TRX h accumulated in the ER but some was also localized to the protein body IIs (PB-IIs). The rice grain quality was dependent on the TRX h accumulation level. Increased TRX h expression resulted in aberrant phenotypes, such as chalky and shriveled features, lower seed weight and lower seed protein content. Furthermore, the accumulation of some seed storage proteins (SSPs) was significantly suppressed and the morphology of the protein bodies (PB-Is and PB-IIs) changed according to the level of TRX h. SSPs, such as 13 kDa prolamin and GluA, were specifically modified via the reducing action of TRX h. These changes led to the activation of the ER stress response, which was accompanied by the expression of several chaperone proteins. Specifically, the ER stress markers BiP4 and BiP5 were significantly up-regulated by an increase in the level of TRX h. These results suggest that changes in the conformation of certain SSPs via the action of recombinant TRX h lead to an induced ER stress response in transgenic rice seeds.     

Thiol status in human sperm

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol-disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence-activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% +/- 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P less than 0.05). However, within the normozoospermic group, no correlation was found between thiol-labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between "swim-up" and "whole semen" preparations.     

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.     

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29–32 kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58 kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58 kDa putative globulin and a 6 kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H₂O₂-oxidized 6 kDa polypeptide, but not the 58 kDa polypeptide by DTT. The solubility of 6 kDa putative albumin, 58 and 19–24 kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58 kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58 kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58 kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes.     

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.     

Glutathione disulfide and S-nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

Mycobacterium tuberculosis resides within alveolar macrophages. These phagocytes produce reactive nitrogen and oxygen intermediates to combat the invading pathogens. The macrophage glutathione (GSH) pool reduces nitric oxide (NO) to S-nitrosoglutathione (GSNO). Both glutathione disulfide (GSSG) and GSNO possess mycobactericidal activities in vitro. In this study we demonstrate that M. tuberculosis thioredoxin system, comprises of thioredoxin reductase B2 and thioredoxin C reduces the oxidized form of the intracellular mycothiol (MSSM) and is able to efficiently reduce GSSG and GSNO in vitro. Our study suggests that the thioredoxin system provide a general reduction mechanism to cope with oxidative stress associated with the microbe’s metabolism as well as to detoxify xenobiotics produced by the host.     

Thiol redox-sensitive seed proteome in dormant and non-dormant hybrid genotypes of wheat

The thiol redox-sensitive and the total proteome in harvest-ripe grains of closely related genotypes of wheat (Triticum aestivum L.), with either a dormant or a non-dormant phenotype, were investigated using hybrid lines of spring wheat double haploid population segregating transgressively, to gain further insight into seed dormancy controlling events. Redox signalling by reactive oxygen species has been shown to play a role in seed dormancy alleviation. Thiol-disulfide proteins are of particular importance in the context of redox-dependent regulation as a central and flexible mechanism to control metabolic and developmental activities of the cells. Here we describe functional proteomic profiling of reversible oxidoreductive changes and characterize in vivo intrinsic reactivity of cysteine residues using thiol-specific fluorescent labelling, solubility-based protein fractionation, two-dimensional electrophoresis, and mass spectrometry analysis in conjunction with wheat EST sequence libraries. Quantitative differences between genotypes were found for 106 spots containing 64 unique proteins. Forty seven unique proteins displayed distinctive abundance pattern, and among them 31 proteins contained 78 unique redox active cysteines. Seventeen unique proteins with 19 reactive modified cysteines were found to have differential post-translational thiol redox modification. The results provide an insight into the alteration of thiol-redox profiles in proteins that function in major processes in seeds and include groups of redox- and stress-responsive, genetic information processing and cell cycle control, transport and storage proteins, enzymes of carbohydrate metabolism, proteases and their inhibitors.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.