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Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

ABSTRACT: Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an ?, ?-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the ?, ?-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin.

SUBMITTER: Chen W

PROVIDER: S-EPMC5623065 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

Chen Wei W Tuladhar Anupama A Rolle Shantelle S Lai Yanhao Y Rodriguez Del Rey Freddy F Zavala Cristian E CE Liu Yuan Y Rein Kathleen S KS

Toxicology and applied pharmacology 20170525

Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrop ...[more]

PMID: 28551108

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Project description:High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.

Project description:Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.

Dataset Information

Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

Publications

Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

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