Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2–C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.

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Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02900-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7072-7082 ◽

Cited By ~ 16

Author(s):

Horacio Reyes-Vivas ◽

Ignacio de la Mora-de la Mora ◽

Adriana Castillo-Villanueva ◽

Lilian Yépez-Mulia ◽

Gloria Hernández-Alcántara ◽

...

Keyword(s):

Giardia Lamblia ◽

Cytotoxic Effect ◽

Triosephosphate Isomerase ◽

Glycolytic Enzyme ◽

Cysteine Residue ◽

Enzyme Inactivation ◽

Dependent Manner ◽

Drug Resistant ◽

Content Type

ABSTRACTGiardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains ofGiardia lambliawas evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites.G. lambliatriosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effectivein vitroagainst drug-resistant and drug-susceptible strains ofG. lamblia.

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ZnO NPs delay the recovery of psoriasis-like skin lesions through promoting inflammation and keratinocyte apoptosis via nuclear translocation of phosphorylated NF-κB p65 and cysteine deficiency

10.21203/rs.3.rs-22538/v1 ◽

2020 ◽

Author(s):

Xuan Lai ◽

Menglei Wang ◽

Yixia Zhu ◽

Xiaoli Feng ◽

Huimin Liang ◽

...

Keyword(s):

Skin Diseases ◽

Nuclear Translocation ◽

Normal Skin ◽

Redox Homeostasis ◽

Skin Lesions ◽

Zno Nps ◽

Inflammatory Skin Diseases ◽

Tnf Α ◽

Jnk Inhibitors

Abstract Background This study aimed to confirm the safety and risk of applying zinc oxide nanoparticles (ZnO NPs) to pathological skin, such as psoriasis-like skin. The majority of previous studies confirmed the safety of applying ZnO NPs to normal skin. However, we know very little about the risks of using sunscreen, cosmetics and topical drugs containing ZnO NPs for individuals with skin diseases. In addition, some studies claimed that ZnO NPs can penetrate normal or pathological skin, and ZnO NPs have frequently been reported to have proinflammatory and lethal effects in vitro. Therefore, it is necessary to evaluate the safety of applying ZnO NPs to pathological skin. Results ZnO NPs passed through gaps between keratinocytes and entered stratum basale of epidermis and dermis in imiquimod (IMQ)-induced psoriasis-like skin lesions. Application of a ZnO NP-containing suspension for 3 connective days delayed the healing of the epidermal barrier; increased the expression levels of inflammatory cytokines; promoted keratinocyte apoptosis and disturbed redox homeostasis. In vitro, ZnO NPs promoted TNF-α, IL-1β and IL-6 secretion and apoptosis of recombinant-human-TNF-α-stimulated HaCaT cells. NF-κB, ERK, p38 and JNK inhibitors blocked ZnO NP-induced inflammation. JSH-23, an inhibitor of the nuclear translocation of p-NF-κB p65, and NAC, an acetylated precursor of L-cysteine, not only inhibited the ZnO NP-induced inflammation but also inhibited apoptosis and cysteine deficiency. Neither erastin nor RSL3 induced p-NF-κB p65 nuclear translocation, but they did reduce cysteine biosynthesis. Additionally, ferropstatin-1, an inhibitor of lipid peroxidation, partially rescued ZnO NP-induced decreases in cell viability and cysteine content. Conclusions ZnO NPs delay the recovery of psoriasis-like skin lesions through promoting inflammation and keratinocyte apoptosis via the nuclear translocation of phosphorylated NF-κB p65 and cysteine deficiency. This work reminds the public that ZnO NPs are not safe for pathological skin, especially in inflammatory skin diseases such as psoriasis, and has revealed a partial mechanism by which ZnO NPs delay the recovery of pathological skin, promoting the appropriate use of ZnO NPs.

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Heterologous Expression of Functional Ptr ToxA

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.4.456 ◽

2000 ◽

Vol 13 (4) ◽

pp. 456-464 ◽

Cited By ~ 37

Author(s):

Robert P. Tuori ◽

Thomas J. Wolpert ◽

Lynda M. Ciuffetti

Keyword(s):

Fusion Protein ◽

Heterologous Expression ◽

Specific Activity ◽

Maximal Activity ◽

Cysteine Residue ◽

Binding Studies ◽

Reading Frame ◽

Pyrenophora Tritici Repentis ◽

Ptr Toxa

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

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Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bj20111993 ◽

2012 ◽

Vol 444 (2) ◽

pp. 227-237 ◽

Cited By ~ 10

Author(s):

Kaushik Saha ◽

Michael E. Webb ◽

Stephen E. J. Rigby ◽

Helen K. Leech ◽

Martin J. Warren ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Quaternary Structure ◽

Specific Activity ◽

Close Association ◽

Physiological Role ◽

Cysteine Residue ◽

In Planta ◽

Iron Sulfur ◽

Tetrapyrrole Metabolism

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe–S] cluster. We determined the cluster to be a [4Fe–4S] type, which quickly oxidizes to a [2Fe–2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys135, is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe–4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe–S cluster or Cys135 retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe–S cluster in planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

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Antioxidant Activity of the Yeast Mitochondrial One-Cys Peroxiredoxin Is Dependent on Thioredoxin Reductase and Glutathione In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01918-08 ◽

2009 ◽

Vol 29 (11) ◽

pp. 3229-3240 ◽

Cited By ~ 50

Author(s):

Darren Greetham ◽

Chris M. Grant

Keyword(s):

Oxidative Stress ◽

Antioxidant Activity ◽

Thioredoxin Reductase ◽

Disulfide Bridge ◽

Cysteine Residue ◽

In Vitro Assays ◽

Redox Partner ◽

Thioredoxin System

ABSTRACT Peroxiredoxins are ubiquitous enzymes which protect cells against oxidative stress. The first step of catalysis is common to all peroxiredoxins and results in oxidation of a conserved peroxidatic cysteine residue to sulfenic acid. This forms an intermolecular disulfide bridge in the case of 2-Cys peroxiredoxins, which is a substrate for the thioredoxin system. 1-Cys Prx's contain a peroxidatic cysteine but do not contain a second conserved cysteine residue, and hence the identity of the in vivo reduction system has been unclear. Here, we show that the yeast mitochondrial 1-Cys Prx1 is reactivated by glutathionylation of the catalytic cysteine residue and subsequent reduction by thioredoxin reductase (Trr2) coupled with glutathione (GSH). This novel mechanism does not require the usual thioredoxin (Trx3) redox partner of Trr2 for antioxidant activity, although in vitro assays show that the Trr2/Trx3 and Trr2/GSH systems exhibit similar capacities for supporting Prx1 catalysis. Our data also indicate that mitochondria are a main target of cadmium-induced oxidative stress and that Prx1 is particularly required to protect against mitochondrial oxidation. This study demonstrates a physiological reaction mechanism for 1-Cys peroxiredoxins and reveals a new role in protection against mitochondrial heavy metal toxicity.

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Parkinson Disease-Linked Parkin Mediates Redox Reactions That Lower Oxidative Stress In Mammalian Brain

10.1101/2020.04.26.062380 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel N. El Kodsi ◽

Jacqueline M. Tokarew ◽

Rajib Sengupta ◽

Nathalie A. Lengacher ◽

Andy C. Ng ◽

...

Keyword(s):

Oxidative Stress ◽

Reductase Activity ◽

Redox Homeostasis ◽

Redox Balance ◽

Oxidative Modification ◽

Reduced Form ◽

Mammalian Brain ◽

Anti Oxidant

SUMMARYWe recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H2O2) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H2O2 levels were unchanged in adult prkn-/- brain, a parkin-dependent reduction of cytosolic H2O2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn-/- mice. Nevertheless, this rise in oxidative stress led to changes in mitochondrial enzyme activities and the metabolism of glutathione in cells and mammalian brain. In parkin’s absence reduced glutathione concentrations were increased including in human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H2O2 reduction and glutathione regeneration. These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance.

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Cardiomyocyte-specific Txnip C247S mutation improves left ventricular functional reserve in streptozotocin-induced diabetic mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00174.2021 ◽

2021 ◽

Author(s):

Nobuhiro Mukai ◽

Yoshinobu Nakayama ◽

Syed Amir Abdali ◽

Jun Yoshioka

Keyword(s):

Oxidative Stress ◽

Molecular Mechanisms ◽

Redox Homeostasis ◽

Disulfide Bridge ◽

Cysteine Residue ◽

Left Ventricular ◽

Contractile Reserve ◽

Wild Type ◽

Littermate Control ◽

In Cells

Underlying molecular mechanisms for the development of diabetic cardiomyopathy remain to be determined. Long-term exposure to hyperglycemia causes oxidative stress, which leads to cardiomyocyte dysfunction. Previous studies established the importance of thioredoxin-Interacting protein (Txnip) in cellular redox homeostasis and glucose metabolism. Txnip is a highly glucose-responsive molecule that interacts with the catalytic center of reduced thioredoxin and inhibits the antioxidant function of thioredoxin. Here, we show that the molecular interaction between Txnip and thioredoxin plays a pivotal role in the regulation of redox balance in the diabetic myocardium. High glucose increased Txnip expression, decreased thioredoxin activities, and caused oxidative stress in cells. The Txnip-thioredoxin complex was detected in cells with overexpressing wild-type Txnip but not Txnip cysteine 247 to serine (C247S) mutant that disrupts the intermolecular disulfide bridge. Then, diabetes was induced in cardiomyocyte-specific Txnip C247S knock-in mice and their littermate control animals by injections of streptozotocin (STZ). Prolonged hyperglycemia up-regulated myocardial Txnip expression in both genotypes. The absence of Txnip's inhibition of thioredoxin in Txnip C247S mutant hearts promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage by diabetes. Stress hemodynamic analysis uncovered that Txnip C247S knock-in hearts have a greater left ventricular contractile reserve than wild-type hearts under STZ-induced diabetic conditions. These results provide novel evidence that Txnip serves as a regulator of hyperglycemia-induced cardiomyocyte toxicities through direct inhibition of thioredoxin, and identify the single cysteine residue in Txnip as a therapeutic target for diabetic injuries.

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Activation of cell-penetrating peptide fragments by disulfide formation

Amino Acids ◽

10.1007/s00726-020-02880-x ◽

2020 ◽

Vol 52 (8) ◽

pp. 1161-1168

Author(s):

Raheleh Tooyserkani ◽

Wojciech Lipiński ◽

Bob Willemsen ◽

Dennis W. P. M. Löwik

Keyword(s):

Targeted Drug Delivery ◽

Disulfide Bridge ◽

Cell Penetrating Peptides ◽

Cysteine Residue ◽

Fluorescent Label ◽

Peptide Fragments ◽

Disulfide Formation ◽

Cell Penetrating ◽

Uptake Activity

Abstract Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.

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Inhibition of calpain-1 stabilizes TCF11/Nrf1 but does not affect its activation in response to proteasome inhibition

Bioscience Reports ◽

10.1042/bsr20180393 ◽

2018 ◽

Vol 38 (5) ◽

Cited By ~ 1

Author(s):

Karolin Nowak ◽

Ramona M. Taubert ◽

Stefanie Haberecht ◽

Simone Venz ◽

Elke Krüger

Keyword(s):

Transcription Factor ◽

Nuclear Translocation ◽

Cultured Cells ◽

Redox Homeostasis ◽

Proteasome Inhibition ◽

Calcium Dependent ◽

Proteotoxic Stress ◽

Chemical Inhibitors ◽

Membrane Bound

Protein degradation is essential to compensate for the damaging effects of proteotoxic stress. To ensure protein and redox homeostasis in response to proteasome inhibition, the cleavage and nuclear translocation of the endoplasmic reticulum (ER)-bound transcription factor TCF11/Nrf1 (NFE2L1) is crucial for the activation of rescue factors including the synthesis of new proteasomal subunits. Even though TCF11/Nrf1 is an essential transcription factor, the exact mechanisms by which it is activated and stabilized are not fully understood. It was previously shown that the calcium-dependent protease calpain-1 interacts with TCF11/Nrf1 and the TCF11/Nrf1 cleavage site is a potential calpain target. Here, we tested the hypothesis that calpain-1 or -2 cleave TCF11/Nrf1. However, we did not find a role for calpain-1 or -2 in the activation of TCF11/Nrf1 after proteasome inhibition neither by using chemical inhibitors nor siRNA-mediated knockdown or overexpression of calpain subunits. Instead, we found that TCF11/Nrf1 is digested by calpain-1 in vitro and that calpain-1 inhibition slows down the degradation of membrane-bound TCF11/Nrf1 by the proteasome in cultured cells. Thus, we provide evidence that calpain-1 is involved in the degradation of TCF11/Nrf1. Furthermore, we confirmed DDI2 as an essential factor for TCF11/Nrf1 activation and demonstrate an undefined role of DDI2 and calpain-1 in TCF11/Nrf1 stability.

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Targeting the Nrf2/ARE Signalling Pathway to Mitigate Isoproterenol-Induced Cardiac Hypertrophy: Plausible Role of Hesperetin in Redox Homeostasis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/9568278 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Prema Velusamy ◽

Thangarajeswari Mohan ◽

Divya Bhavani Ravi ◽

S. N. Kishore Kumar ◽

Ashokkumar Srinivasan ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Antioxidant Defense ◽

Nuclear Translocation ◽

Redox Homeostasis ◽

Cardiac Remodelling ◽

Antioxidative Status ◽

Matrix Deposition

Cardiac hypertrophy is the underlying cause of heart failure and is characterized by excessive oxidative stress leading to collagen deposition. Therefore, understanding the signalling mechanisms involved in excessive extracellular matrix deposition is necessary to prevent cardiac remodelling and heart failure. In this study, we hypothesized that hesperetin, a flavanone that elicits the activation of Nrf2 signalling and thereby suppresses oxidative stress, mediated pathological cardiac hypertrophy progression. A cardiac hypertrophy model was established with subcutaneous injection of isoproterenol in male Wistar rats. Oxidative stress markers, antioxidant defense status, and its upstream signalling molecules were evaluated to discover the impacts of hesperetin in ameliorating cardiac hypertrophy. Our results implicate that hesperetin pretreatment resulted in the mitigation of oxidative stress by upregulating antioxidant capacity of the heart. This curative effect might be owing to the activation of the master regulator of antioxidant defense system, known as Nrf2. Further, analysis of Nrf2 revealed that hesperetin enhances its nuclear translocation as well as the expression of its downstream targets (GCLC, NQO1, and HO-1) to boost the antioxidative status of the cells. To support this notion, in vitro studies were carried out in isoproterenol-treated H9c2 cells. Immunocytochemical analysis showed augmented nuclear localization of Nrf2 implicating the action of hesperetin at the molecular level to maintain the cellular redox homeostasis. Thus, it is conceivable that hesperetin could be a potential therapeutic candidate that enhances Nrf2 signalling and thereby ameliorates pathological cardiac remodelling.

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