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Effectors of thioredoxin reductase: Brevetoxins and manumycin-A
Anupama Tuladhar1, Robert J Hondal2, Ricardo Colon1
1Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, United States.
Abstract:
The activities of two effectors, brevetoxin (PbTx) and manumycin-A (Man-A), of thioredoxin reductase (TrxR) have been evaluated against a series of fourteen TrxR orthologs originating from mammals, insects and protists and several mutants. Man-A, a molecule with numerous electrophilic sites, forms a covalent adduct with most selenocystine (Sec)-containing TrxR enzymes. The evidence also demonstrates that Man-A can form covalent adducts with some non-Sec-containing enzymes. The activities of TrxR enzymes towards various substrates are moderated by Man-A either positively or negatively depending on the enzyme. In general, the reduction of substrates by Sec-containing TrxR is inhibited and NADPH oxidase activity is activated. For non-Sec-containing TrxR the effect of Man-A on the reduction of substrates is variable, but NADPH oxidase activity can be activated even in the absence of covalent modification of TrxR. The effect of PbTx is less pronounced. A smaller subset of enzymes is affected by PbTx. With a single exception, the activities of most of this subset are activated. Although both PbTx variants can react with selenocysteine, a stable covalent adduct is not formed with any of the TrxR enzymes. The key findings from this work are (i) the identification of an alternate mechanism of toxicity for the algal toxin brevetoxin (ii) the demonstration that covalent modification of TrxR is not a prerequisite for the activation of NADPH oxidase activity of TrxR and (iii) the identification of an inhibitor which can discriminate between cytosolic and mitochondrial TrxR.
Insights
Manumycin-A (Man-A) modifies thioredoxin reductase (TrxR) enzymes, affecting their activity and revealing new toxicity mechanisms for brevetoxin (PbTx). This study identifies inhibitors that distinguish between cytosolic and mitochondrial TrxR.
Area of Science:
- Biochemistry
- Enzymology
- Toxicology
Background:
- Thioredoxin reductase (TrxR) is a crucial enzyme in cellular redox homeostasis.
- Understanding TrxR regulation and inhibition is vital for developing targeted therapies.
- Effectors like brevetoxin (PbTx) and manumycin-A (Man-A) offer insights into TrxR function.
Purpose of the Study:
- To investigate the activities of PbTx and Man-A on a diverse range of TrxR orthologs and mutants.
- To elucidate the mechanisms by which Man-A interacts with both selenocysteine-containing and non-selenocysteine-containing TrxRs.
- To identify novel inhibitors capable of discriminating between different TrxR isoforms.
Main Methods:
- Enzyme activity assays were performed using fourteen TrxR orthologs from various species and several mutants.
- Covalent adduct formation was analyzed to understand effector-enzyme interactions.
- Substrate reduction and NADPH oxidase activity were measured to assess enzyme modulation.
Main Results:
- Man-A forms covalent adducts with most selenocysteine (Sec)-containing TrxRs and some non-Sec-containing TrxRs, altering their activities.
- Man-A generally inhibits substrate reduction in Sec-containing TrxRs while activating NADPH oxidase activity.
- PbTx affects a smaller subset of TrxRs, primarily activating their activities without stable covalent adduct formation.
- Crucially, covalent modification of TrxR is not required for NADPH oxidase activity activation.
Conclusions:
- Man-A exhibits varied effects on TrxR activity, including covalent modification and modulation of NADPH oxidase activity.
- Brevetoxin (PbTx) presents an alternative toxicity mechanism independent of stable covalent adducts with TrxR.
- This research identifies a novel inhibitor capable of differentiating between cytosolic and mitochondrial TrxR, paving the way for targeted therapeutic strategies.
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