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 α, β-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 α, β-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.

Keywords: Brevetoxin; Karenia brevis; Thioredoxin; Thioredoxin reductase.

Figure 1

Structures of the two most abundant brevetoxins having the B-type backbone

Figure 2

Electron flow of mammalian TrxR/Trx system. Reduced sites are shown in red.

Figure 3

The effect of brevetoxin on TrxR/Trx system, TrxR, Grx and GPx. (A/B) The effect of PbTx-2 (A) or PbTx-3 (B) on the TrxR/Trx system in the fluorescent insulin reduction assay. Rat TrxR and human Trx pre-reduced with NADPH (0.35 mM) for 30 min followed by incubation with brevetoxins for 1 h prior to addition of insulin substrate. (C) The effect of PbTx-2 on Grx. (D) The effect of PbTx-2 on GPx.

Figure 4

(A/B) Rat TrxR was pre-reduced with NADPH (0.10 mM) for 30 min either with (A) or without (B) PbTx-2 (18 μM), followed by addition of DTNB (2 mM). (C) Truncated human TrxR was pre-reduced with NADPH (0.10 mM) for 30 min in the presence of PbTx-2 (18 μM), followed by DTNB (2 mM). D. Rat TrxR was pre-reduced with NADPH (0.10 mM) for 30 min followed by incubation with curcumin (22 μM, 30 min) or curcumin (22 μM, 30 min) then PbTx-2 (22 μM, 30 min) or PbTx-2 (22 μM, 30 min) then curcumin (22 μM, 30 min), prior to addition of DTNB (2 mM). Data are expressed as % of control at 60 min.

Figure 5

Release of fluorescent reporter from Sel-green probe upon incubation with TrxR in the presence and absence of PbTx-2 compared to standards of L-selenocysteine (3 μM and 6 μM).

Figure 6

A. 24 hr dose response curve for human lymphoblast cells (GMO2125) in the presence of PbTx-2. EC50 for PbTx-2 is 2.4 μM and 2.4 μM in the presence of 100 μM trolox. B. Cell viability (MTT), cellular glutathione (DTNB), selenol content (Sel green) and lipid peroxidation (TBARS) for PbTx-2 (1 μg/mL) treated and PbTx-2 (1 μg/mL) treatment simultaneous with trolox (100 μg/mL). *Indicates a statistically significant difference from the control (p ≤ 0.05).

Figure 7

Electron flow in alkylated TrxR.

Scheme 1

Reaction of Sel-Green probe with selenocysteine.