Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

Background: Parkinson's disease (PD) is a multifactorial neurodegenerative disease with pathogenic mechanisms traceable to oxidative damage and mitochondrial dysfunction. Rotenone, a chemical compound commonly found in pesticides, has been found to inhibit mitochondrial complex-I and initiate PD-like symptoms in mammals and several invertebrates. Virgin Coconut Oil (VCNO) obtained from the coconut fruit has been found to possess anti-oxidative and anti-inflammatory properties. Objectives: The present study evaluated the effect of VCNO on rotenone-induced Parkinsonism in fruit flies- Drosophila melanogaster (D. melanogaster). Methods: Canton special (CS) strains of D. melanogaster, aged between 1 to 3 days were orally exposed for 7 days to 0, 250, 500 and 750 μM rotenone diet for toxicity assay, and 0, 2.5, 5 and 10 % w/w VCNO diet for longevity assay. Thereafter, 5 % VCNO diet was selected for evaluation against 500 μM rotenone. Subsequently, behavioural test (negative geotaxis), markers for redox status and enzyme activities were evaluated. Results: The results showed that rotenone induced toxicity in the flies, while VCNO increased the lifespan of D. melanogaster in a dose-dependent manner. In addition, VCNO ameliorated rotenone-induced locomotor deficits, elevated MDA, as well as the depleted GSH levels. It also mitigated the inhibited activities of SOD, CAT and ATPase in the flies. Conclusions: VCNO protected D. melanogaster against rotenone-induced toxicity by extending longevity, preventing locomotor deficits and reducing oxidative stress.

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A Novel Approach for In Vivo Measurement of Red Cell Redox Status

Blood ◽

10.1182/blood.v116.21.2036.2036 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2036-2036 ◽

Cited By ~ 1

Author(s):

Xiuling Xu ◽

Katharina von Loehneysen ◽

Deborah Noack ◽

Andrew Vu ◽

Jeff S. Friedman

Keyword(s):

Fluorescence Emission ◽

Redox Status ◽

Red Cells ◽

Cysteine Residues ◽

Excitation Wavelength ◽

Red Cell ◽

Reducing Conditions ◽

Cell Redox Status

Abstract Abstract 2036 Maintenance of a reducing redox balance is a critical physiologic function of red cell metabolic machinery. Perturbation of this balance, whether inherited or acquired, is found in a variety of red cell pathologies. Methods for evaluation of red cell redox status include direct approaches such as determining glutathione (GSH, GSSG) levels, and indirect approaches such as measuring fluorescence of oxidation sensitive dyes. Here we describe an alternative method for evaluation of red cell redox status that can be used in vivo and in real-time assays. Engineered variants of GFP possessing two solvent accessible cysteine residues function as molecular redox sensors with distinct fluorescence characteristics. Excitation spectrum shifts upon the oxidation of cysteine residues forming a disulfide. A higher ratio of fluorescence when comparing excitation at 405nm versus 488nm indicates rising levels of oxidized GFP and a shift in cellular redox status. To validate redox GFPs in erythroid cells, we first performed in vitro assays with MEL cells over-expressing several related GFP sensors (ro-GFPs), selecting the brightest molecule (roGFP2) for further study. The sensor function of roGFP2 in MEL cells was verified by stimulation with exogenous oxidant (1mM H202) or reductant (10 mM DTT) as shown in the figure below. In order to create a physiologic in vivo model for study of red cell redox status, transgenic mice expressing roGFP2 specifically in the erythroid lineage were generated. roGFP2 expressing red cells demonstrate the expected shift in fluorescence upon exposure to H202 or t-butyl peroxide in a short-term assay. In vivo, we have measured red cell lifespan (using biotin-labeling) in roGFP2 transgenic animals to follow redox status of red cells as a function of cell age. Expression of roGFP2 has no effect on red cell survival. Interestingly, when comparing old red cells (age > 50days) with younger cells (age < 50days), a shift in GFP fluorescence ratio indicating that a higher fraction of the sensor is oxidized in the aged cells was observed. This observation is consistent with the hypothesis that metabolic changes, in particular a decline in ability to reduce oxidative damage, contribute to red cell senescence. We are generating several murine strains with defined red cell defects also expressing roGFP2 in order to assess the role of changes in intra-erythrocyte redox status in a range of pathologic conditions. In vitro and in vivo assays using roGFP2 transgenic cells/mice are in process to determine the potential utility of this system as a screen for hematoxicity of drugs and other compounds. Figure 1 Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Figure 1. Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Disclosures: No relevant conflicts of interest to declare.

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Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster

Redox Biology ◽

10.1016/j.redox.2015.06.001 ◽

2015 ◽

Vol 5 ◽

pp. 328-339 ◽

Cited By ~ 34

Author(s):

Amos O. Abolaji ◽

Jean P. Kamdem ◽

Thiago H. Lugokenski ◽

Ebenezer O. Farombi ◽

Diogo O. Souza ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Redox Status ◽

Vinylcyclohexene Diepoxide

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Basal Glutathionylation of Na,K-ATPaseα-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/9092328 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Vladimir A. Mitkevich ◽

Irina Yu. Petrushanko ◽

Yuri M. Poluektov ◽

Ksenia M. Burnysheva ◽

Valentina A. Lakunina ◽

...

Keyword(s):

Acute Hypoxia ◽

Viral Proteins ◽

Redox Status ◽

Cysteine Residues ◽

Potassium Ions ◽

X Ray ◽

Pig Kidney ◽

Sodium And Potassium ◽

Enzyme Biosynthesis

Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPaseα-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPaseα-subunit; however, basal glutathionylation ofα-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPaseα-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPaseα-subunit and provide evidence supporting the view that this modification is cotranslational.

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A novel approach for predicting protein S-glutathionylation

BMC Bioinformatics ◽

10.1186/s12859-020-03571-w ◽

2020 ◽

Vol 21 (S11) ◽

Cited By ~ 1

Author(s):

Anastasia A. Anashkina ◽

Yuri M. Poluektov ◽

Vladimir A. Dmitriev ◽

Eugene N. Kuznetsov ◽

Vladimir A. Mitkevich ◽

...

Keyword(s):

Disulfide Bonds ◽

Prediction Method ◽

Protein S ◽

Redox Status ◽

Cysteine Residue ◽

Cysteine Residues ◽

Amino Acid Residues ◽

Novel Approach ◽

Positive Dataset ◽

Unknown Structure

Abstract Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

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