Effectors of thioredoxin reductase: Brevetoxins and manumycin-A

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 selenocytine, 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.

Graphical abstract

1. Introduction

The thioredoxin system, comprised of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, is a major cellular antioxidant system that functions to maintain redox homeostasis (Matsuzawa, 2017; Mahmood et al., 2013; Lu and Holmgren, 2012; Holmgren and Lu, 2010; Arner, 2009.). This is accomplished in part through the activity of Trx, which reduces target protein disulfide bridges by thiol-disulfide exchange. The cellular pool of reduced Trx is maintained by TrxR, which accepts reducing equivalents from NADPH and transfers these reducing equivalents through a series of redox centers to the oxidized form of Trx (Fig. 1).

Fig. 1.

Electron flow from NADPH to protein disulfide bridges, via TrxR and Trx. Trx reduces many proteins and is returned to the active reduced state by TrxR in a NADPH dependent reaction. The C-terminal redox center (in the box) in high M_r TrxR can be composed of two cysteine residues or a cysteine and a selenocysteine.

TrxR is present in all organisms and numerous orthologs have evolved (Lu and Holmgren, 2014). TrxR orthologs differ in size and the number and sequence of redox centers. These variations form the basis of the classification systems. TrxRs from lower eukaryotes and prokaryotes are low molecular weight dimers of ~35 kDa subunits that utilize two redox centers: a non-covalently bound flavin adenine dinucleotide (FAD) and an N-terminal dithiol/disulfide (Cys-Ala-Thr-Cys) (Williams et al., 2000).

The high-M_r TrxRs (present in numerous animals, including vertebrates, invertebrates, insects and protists) are composed of two high molecular weight (M_r ~55 kDa) subunits arranged such that the reducing equivalent of NADPH is transferred via FAD and the N-terminal dithiol to an additional C-terminal redox center of the other monomer (Bauer et al., 2003). This C-terminal redox center is a flexible extension of 16-amino acids in the mammalian enzyme, not present in the low-M_r enzymes, whose sequence varies according to the source. Mammalian TrxR has three isoforms: TrxR-1 (cytosolic TrxR), TrxR-2 (mitochondrial TrxR) and thioredoxin glutathione reductase (testicular TGR).

TrxR from higher organisms can be further classified into two groups based on the relative positions of the redox active residues making up the C-terminal redox center (Table 1). Type I TrxR contains adjacent redox residues: either Cys-Sec (Type 1a), or Cys-Cys (Type 1b). Type II TrxR have the redox cysteines separated by four amino acids (Cys-Gly-Gly-Gly-Lys-Cys-Gly) as in Plasmodium falciparum. Despite the sequence disparity in the C-terminal redox center, these TrxR enzymes have a similar mechanism of action.

Table 1:

Classification of TrxR on the basis of C-terminal redox center

TrxR Type	Representative	C-terminal motif	Dependent on C- terminal redox center to reduce DTNB?
1a	mammalian TrxR-1 (cytosolic)	GCUG	Yes
	mammalian TrxR-2 (mitochondrial)	GCUG	No
	mammalian TGR (testes specific)	GCUG	No
1b	Caenorhabditis elegans (mitochondrial)	GCCG	No
	Drosophila melanogaster (cytosolic/mitochondrial)	SCCS	No
II	Plasmodium falciparum	CGGGKCG	No

In its reduced form, the Sec residue of Type Ia TrxR is susceptible to alkylation with electrophiles. Numerous inhibitors of Type Ia TrxR act by reacting with and inactivating the C-terminal redox center. Some are α, β-unsaturated carbonyls such as quinones (Cai et al., 2012; Citta et al., 2014), curcumin (Jayakumar et al., 2016) and 4-hydroxy-2-nonenal (Ansari et al., 2017) all of which can function as electrophiles in a 1,4 conjugate addition or Michael reaction (Fig. 2), forming a covalent adduct, which results in the irreversible inhibition of Type Ia TrxR.

Fig. 2.

Mechanism of Michael addition. Nuc represents the nucleophilic selenoate (RSe⁻).

The brevetoxins (PbTxs) are polycyclic polyether ladder compounds produced by the Florida red tide dinoflagellate, Karenia brevis (Fig. 3). Among the most abundant of the brevetoxins are PbTx-2 and PbTx-3, each composed of 11 fused rings with an α, β-unsaturated aldehyde or allylic alcohol at the K-ring side chains, respectively. Brevetoxins are neurotoxins that bind to site 5 of the voltage-gated sodium channel of excitable membranes resulting in sodium influx into the cell and membrane depolarization, thus affecting both the central nervous system and skeletal muscle (Jeglitsch et al., 1998). This is believed to be the principle mechanism of the neurotoxic effects. However, several studies have reported increases in indicators of oxidative stress in marine animals (Walsh et al., 2015; Walsh et al., 2010) that have been exposed to red tide as well as brevetoxin exposed cells lines (Walsh et al., 2009; Sayer et al., 2005; Murrell and Gibson, 2009)

Fig. 3.

The structures of the brevetoxins and Manumycin-A

Recently we have shown that the Florida red tide toxin, brevetoxin (PbTx-2) inhibits Trx reduction by mammalian TrxR-1. Nonetheless, brevetoxin proved to be unique among TrxR inhibitors. While brevetoxin (20 μM) was found to inhibit Trx reduction, at the same time, it activates reduction of the low molecular weight disulfide substrate DTNB [5,5’-dithio-bis-(2-nitrobenzoic acid)] by 2.5-fold (Chen et al., 2017). This dual effect on TrxR had not been previously observed. This finding is indicative of an alternate mechanism of toxicity, which may broaden the range of effects of PbTx.

We reasoned that molecules of similar size and functionality may also act on TrxR-1 in the same fashion as brevetoxin. We therefore screened a series of molecules for their effect on rat TrxR-1 (rTrxR-1). Through this screening process, we discovered that the cytotoxic manumycin A (Man-A, Fig. 3) behaves as a typical TrxR inhibitor towards rTrxR-1, inhibiting both Trx and DTNB reduction (Tuladhar and Rein, 2018). Man-A is a bacterial secondary metabolite and farnesyl transferase (FTase) inhibitor (Hara et al., 1993). The tumoricidal activity of manumycin was initially attributed to the inhibition of FTase preventing activation of the oncogenic protein Ras. However, it soon became apparent that the cytotoxicity of Man-A included Ras independent pathways. Several studies report the induction of reactive oxygen species (ROS) or more specifically, superoxide radical anion (O₂⁻) in Man-A treated cells and tumors (Ahmad et al., 2015; Ahmad et al., 2011; Carey et al., 2015; Chang et al., 2014; Dixit et al., 2009; Pan et al., 2005; Sears et al., 2008; She et al., 2006; Yu et al., 2012; Zhang et al., 2016).

Herein, we report the unique behavior of, these compounds towards rat TrxR-1 (rTrxR-1, Type 1a), mouse mitochondrial TrxR-2 (mTrxR-2, Type 1a), D. melanogaster TrxR (DmTrxR, Type 1b), and P. falciparium TrxR (PfTrxR, Type II). The Type 1a TrxR in this study (rTrxR-1) is highly dependent upon Sec to reduce DTNB and other small molecule substrates (Zhong and Holmgren, 2000; Lothrop et al., 2014). This characteristic distinguishes the cytosolic enzyme from Type 1b and Type II TrxRs and may help explain how the α, β-unsaturated carbonyl compounds used in this study differentially affect the activity of the various enzymes in Table 1. The abbreviations for all of the enzymes are derived as follows: one or two letters designating the name of species from which the enzyme originated-TrxR-1 for cytosolic, TrxR -2 for mitochondrial, one letter amino acid code for the sequence of the C-terminal redox center listed in the Tables but only the last four amino acids are listed in the text. The designation “Δ” followed by a number denotes a truncated enzyme with the number amino acids missing from the C-terminus.

We also examined how the presence or absence of Sec in the enzymes listed in Table 1 altered the sensitivity of TrxR towards Man-A, PbTx-2, and PbTx-3 by testing mutant enzymes prepared by protein semisynthesis (Eckenroth et al., 2006, 2007; Snider et al., 2014; Lothrop et al., 2014). In addition, for the Type II PfTrxR, we were able to construct chimeric enzymes in which we replaced the Type II C-terminal redox center with either a Type 1a or Type 1b redox center to test how the identity of the redox center impacts the way in which Man-A, PbTx-2, and PbTx-3 changed the activity of the enzyme.

2. Materials and Methods

2.1. General experimental details

Brevetoxins, PbTx-2 and PbTx-3 were purified from cultures of K. brevis according to published protocols (Baden et al., 1981). Papain, methyl methanethiosulfonate, N–benzoyl–L–arginine-p-nitroanilide, immobilized TCEP (Tris [2–carboxyethyl] phosphine hydrochloride), cystamine were purchased from Thermo Scientific Co. The Sel-green probe was synthesized according to published methods (Zhang et al., 2015). Manumycin A was purchased from BioViotica and used without further purification. Fluorescent insulin reduction assay kit and rat recombinant TrxR-1 were purchased from Cayman Chemical. All other TrxR enzymes and mutants were prepared as previously described (Eckenroth et al., 2006, 2007; Snider et al., 2014; Lothrop et al., 2014). All other reagents were purchased from Sigma-Aldrich or Fisher Scientific and used without further purification. The insulin reduction assay was performed according to the manufacturer's instructions with noted exceptions below. UV/Visible and fluorescence measurements were performed in 384 well microplates using a Synergy® 2 (BioTek Instrument, Inc.) or an Infinite® Ml000 PRO (Tecan Group Ltd.) microplate reader. All assays were performed in triplicate. Results represent an average of three trials ± standard deviation. Error limits on graphs represent the standard deviation of three trials.

2.2. Reaction of brevetoxins and Man-A with L-selenocystine

An aqueous stock solution of L-selenocystine (500 μL, 156 μM) was reduced with 1.5 equivalent of immobilized TCEP (>8 μmol/mL) for 1 hour. After reduction, the immobilized TCEP was removed by centrifugation at 14,000 X g, for 10 minutes. The reduced L-selenocystiene (20 μL, 312 μM) was incubated with PbTx-3 or Man-A (10 μL, 625 μM in 25 % DMSO or MeOH) for 1 hour in 50 mM Tris-HCl, 1 mM EDTA pH 7.5. The mixture was incubated for an additional 1 hour at room temperature. Sel-green (20 μL, 100 μM in 50 mM Tris-HCl, 1 mM EDTA pH 7.5 was added to the reduced L-selenocystiene / PbTx-3 or Man-A mixture (80 μL) to initiate the reaction. Final concentrations: 10 μM TrxR; 20 μM PbTx-3 or Man-A; 20 μM Sel-green. The fluorescence was monitored at λex/λem= 370 nm/510 nm every 5 minutes for 1 hour.

2.3. DTNB reduction assay

The assay was performed in 384 well black flat bottom plates in a final volume of 100 μL. TrxR enzymes (6.4 nM or 7.04 nM) were reduced with NADPH (102 μM or 117 μM) in assay buffer (50 mM Tris-HCl, 1 mM EDTA pH 7.5) for 30 minutes at room temperature. PbTx-2 or Man-A (0.625 mM in 25 % DMSO or 56% MeOH) was added for a concentration of 25 μM. The mixture was incubated for an additional thirty minutes at room temperature. DTNB (20 μL, 10 mM in 0.1 M sodium phosphate and 1 mM EDTA, pH 8.0) was added to the TrxR/ PbTx-2 or Man-A mixture (80 μL) to initiate the reaction. Final concentrations: 5.12 nM or 5.6 nM TrxR; 20 μM test compound; 2 mM DTNB. The reduction of DTNB was monitored by measuring absorbance of the product TNB⁻ at 412 nm every 5 minutes for 1 hour. The DTNB competition assay was performed as described above with brevetoxin (20 μM) added first and incubated for 30 minutes followed by the addition of Man-A (5, 1.8, 0.5, 0.05 and 0.005 μM).

2.4. Gel filtration of brevetoxin or Man-A and TrxR mixture to test for reversibility

TrxR enzymes (80 nM) were reduced with NADPH (1.34 mM) in assay buffer (50 mM Tris-HCl, 1 mM EDTA pH 7.5) for 30 minutes at room temperature. The reduced TrxR was divided into three aliquots viz. control (TrxR/no PbTx-2 or Man-A), TrxR/ PbTx-2 or Man-A and TrxR/ PbTx-2 or Man-A not subjected to gel filtration. A solution of PbTx-2 or Man-A (11 μL, 680 μM PbTx-2 in 56 % MeOH or 140 μM in 25% DMSO) was added to the TrxR samples (280 μL) for concentrations of Man-A (2.5 μM for rTrxR-1 and 5.5 μM for all others) or PbTx-2 (26.7 μM). The control sample was substituted with an equal volume of 25% DMSO or 56% MeOH. Samples were incubated for 1 hour at ambient temperature, after which they were passed through a Micro Bio-spin P-6 gel column of MW limit of 6000 (Bio-Rad) to remove unbound PbTx-2 or Man-A. All samples (280 μL) were re-reduced with NADPH (20 μL, 1.34 mM) for 30 minutes. Finally, the DTNB assay was performed as described above by adding 20 μL of 10 mM DTNB to 80 μL of enzyme/ Man-A or PbTx-2 mixture and the absorbance was monitored at 412 nm every 5 minutes for 1 hour. Final concentrations: 5.2 nM TrxR; 20 μM PbTx-2, 2 μM Man-A for rTrxR-1, 4.2 μM Man-A for TrxR-2 and mutants, 20 μM Man-A for Dm and Pf enzymes.

2.5. Hydrogen peroxidase activity

The assay was performed in a 384 well black flat bottom plates in a final volume of 100 μL. TrxR enzymes (62.5 nM) were reduced with NADPH (200 μM) in the presence of Man-A or PbTx-2 (48 μM) in assay buffer (50 mM Tris-HCl, 1 mM EDTA pH 7.5) for 15 minutes. An uncompromised C-terminal Sec is required for activity. A solution of hydrogen peroxide (20 μL, 250 mM in assay buffer) was added to the enzyme/Man-A or PbTx-2 solution (80 μL). The consumption of NADPH was monitored by measuring absorbance at 340 nm for 10 minutes. NADPH concentrations were calculated from a calibration curve of NADPH of various concentrations (0, 50 μM, 100 μM, 250 μM and 500 μM) NADPH in 50 mM Tris-HCl, 1 mM EDTA pH 7.5. Final concentrations: 50 nM TrxR; 38.5 μM test compound; 50 mM H₂O₂. The potent TrxR inhibitor auranofin (38.5 μM) was used as a control (data not shown).

2.6. TrxR/Trx insulin reduction

The assay was performed in 384 well black flat bottom plates in a final volume of 100 μL. A C-terminal redox center is required for activity. Rat TrxR-1 (125 nM) was pre-reduced for 30 min with NADPH (335 μM) in the presence of PbTx-2 or Man-A (25 μM) in assay buffer (0.2 mg/mL BSA, 50 mM Tris-Cl, 1 mM EDTA pH 7.5) for the concentrations of 125 nM TrxR, 25 μM PbTx-2 or Man-A. Human Trx (1 μM) was added for a Trx concentration of 125 nM. This solution was incubated for an additional 30 minutes. Fluorescent substrate (20 μL, 0.4 mg/mL, eosin labeled bovine insulin) was added to the enzyme/ PbTx-2 or Man-A solution (80 μL) to initiate the reaction. Final concentrations: 100 nM TrxR; 20 μM PbTx-2 or Man-A; 100 nM hTrx-1. The fluorescence was monitored at λex/λem= 520nm/545 nm every 5 minutes for 1 hour. Insulin reduction with mTrxR-2 (CGUG), PfTrxR (GCUG) and DmTrxR (SCUG) was performed as described above except the PfTrxR (CUG) and DmTrxR (SCUG) and hTrx concentrations were doubled.

2.7. Modified papain assay with Man-A

2.7.1. Preparation of modified papain-S-SCH3 (~0.6 mg / ml)

A stock solution of papain (1.5 mL, 26.4 mg / ml) was added to a solution of cysteine (0.6 mM, 28.5 mL in 20 mM Na₃PO₄, 1 mM EDTA pH 6.7 buffer). The solution was incubated at room temperature for 30 min. After which methyl methanethiosulfonate (11 μL, 117 μmol) was added and the reaction mixture was kept in ice for 4 hr. The mixture was then dialyzed (Mr cutoff 6000- 8000) against 1L of 5 mM sodium acetate buffer, containing 50 mM NaCl, pH 4.5 overnight at room temperature. The resulting modified papain is diluted with equal volume of 40 mM Na₃PO₄, 2 mM EDTA pH 7.6 buffer resulting in a final concentration of modified papain of 0.6 mg/mL.

2.7.2. Modified papain assay

To determine if Man-A inhibits Trx, the modified papain assay was performed according to literature methods (Singh et al., 1995) in 384 well black flat bottom plate in a final volume of 250 μL. The thioredoxin (hTrx-1) was reduced with a large (~50-fold) excess of immobilized TCEP (>8 μmol/mL) in 50 mM Tris-HCl, 1mM EDTA pH 7 buffer for 1 hr. The immobilized TCEP was removed by centrifugation at 14, 000 X g, for 10 minutes. The reduced thioredoxin (52.6 μM) was incubated with Man-A (100 μM) for 15 minutes on ice. To the hTrx-1/ Man-A solution (50 μL) were added solutions of modified papain-S-SCH₃ (98 μL, ~0.6 mg / mL), cystamine (15 μL, 4 mM), and finally N-benzoyl–L–arginine–p–nitroanilide (87 μL, 6.89 mM) in 50 mM THAM, 1 mM EDTA pH 6.3 for a final volume of 250 μL. Final concentrations: 10.5 μM Trx; 20 μM Man-A; 0.24 mM cystamine; 2.4 mM N-benzoyl-L–arginine–p–nitroanilide. The absorbance was measured at 410 nm at 5 minute intervals for 1 hour.

2.8. Selenocystine reduction by rTrxR-1 and mTrxR-2 variants

The assay was performed in a 384 well black flat bottom plates in a final volume of 100 μL. TrxR enzymes (25 nM) were reduced with NADPH (251 μM) in the presence of Man-A or PbTx-2 (48 μM) in assay buffer (in 50 mM Tris-HCl, 1 mM EDTA pH 7.5) for 15 minutes. The reaction was initiated by the addition of L-selenocy stine (20 μL, 599 μM in assay buffer) to the enzyme test compound mixture (80 μL). The consumption of NADPH was monitored by measuring absorbance at 340 nm for 10 minutes. NADPH concentrations were calculated from a calibration curve of NADPH. Final concentrations: 20 nM TrxR; 38.5 μM Man-A or PbTx-2; 120 μM selenocystine. The potent TrxR inhibitor auranofin (38.5 μM) was used as a control (data not shown).

2.9. NADPH oxidase by TrxR

The assay was performed in 384 well black flat bottom plates in a final volume of 100 μL. NADPH (7.5 μL, 2.68 mM or 5.36 mM for all Dm and Pf enzymes) in assay buffer (50 mM Tris-HCl, 1 mM EDTA pH 7.5) was added to a solution (92.5 μL) of rat recombinant TrxR-1 or mTrxR-2 or mutants (0.22 μM or 0.43 μM for all Dm and Pf enzymes) and Man-A or PbTx-2 (42 μM) in assay buffer. The consumption of NADPH was monitored at 340 nm for 30 minutes. NADPH concentrations were calculated from a calibration curve of NADPH. Final concentrations: TrxR (0.20 μM), Man-A or PbTx-2 (38.5 μM), NADPH (200 μM). For TrxR-1 the final Man-A concentration was 62.5 μM. NADPH mol min/mol-enzyme was calculated from the standard curve of NADPH.

3. Results

3.1. Reaction of brevetoxins and Man-A with L-selenocysteine

Numerous electrophiles inhibit mammalian TrxR by alkylating the C-terminal Sec. These electrophiles are mainly α, β-unsaturated carbonyl compounds that irreversibly react with TrxR. Two reactive sites are present in PbTx-2: the α, β-unsaturated aldehyde on the K-ring and the α, β-unsaturated A-ring lactone which is also present in PbTx-3. Inspection of the Man-A structure reveals numerous potential sites of reactivity. In addition to the epoxide, any of the four mono- or poly unsaturated carbonyl groups of Man-A may alkylate the Sec of TrxR. The selenol selective probe Sel-green was used to evaluate the reactivity of PbTx-3 and Man-A with Sec. The strongly nucleophilic, reduced selenol undergoes nucleophilic aromatic substitution with the probe, which then releases the fluorophore as shown in Fig. 4. When Sec is alkylated it will not react with the probe and fluorescence will be suppressed. Fig. 4 shows that the release of fluorescent reporter by Sec is inhibited in the presence of Man-A and PbTx-3.

Fig 4.

The reaction of PbTx-3 and Man-A with L-selenocysteine prevents the release of fluorescent reporter.

3.2. Reduction of 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB).

The effect of PbTx-2/3 and Man-A at 20 μM on DTNB reduction by TrxRs was examined. Table 2 shows the initial rates (V₀) of DTNB reduction for each enzyme and rates in the presence of the compounds tested relative to the respective controls. The brevetoxins either enhanced or had no effect on the rate of DTNB reduction by TrxRs. The most strongly activated enzyme was the “dead tail” mutant mTrxR-2 (GSSG), whose rate of DTNB reduction was enhanced by 9 or 10-fold by PbTx-2 and -3 respectively. Among the PfTrxR series of enzymes, only two were significantly activated: the chimeric PfTrxR (GCUG), having a Type Ia C-terminal redox center and the “dead tail” mutant PfTrxR (GKSG). Other enzymes were activated by 2 to 4-fold. Man-A either inhibited or enhanced DTNB reduction depending on the enzyme.

Table 2.

Initial rates (V₀) of DTNB reduction (mol TNB /min-mol TrxR) by various TrxR enzymes and initial rates in the presence of the test compounds relative to control.

Enzyme		V0 Control	Fold change in V0 relative to control
			PbTx-2	PbTx-3	Man-A
1	rTrxR-1 (GCUG)	634 ± 47.2	2.61 ± 0.48**	6.04 ± 1.10**	0.05 ± 0.05**
2	mTrxR-2 (GCUG)	785 ± 11.0	1.41 ± 0.07**	3.16 ± 0.23**	1.48 ± 0.07**
3	mTrxR-2 (GCCG)	249 ± 23.2	4.42 ± 0.85**	3.99 ± 0.40**	4.54 ± 0.43**
4	mTrxR-2 (GSSG)	226 ± 17.0	9.08 ± 0.39**	10.08 ± 0.78**	4.86 ± 0.19**
5	mTrxR-2 (Δ8)	995 ± 65.5	2.61 ± 0.15**	2.29 ± 0.08**	2.45 ± 0.16**
6	DmTrxR (SCUG)	70.4 ± 2.25	2.07 ± 0.10**	2.97 ± 0.10**	0.09 ± 0.06**
7	DmTrxR (SCCS)	34.5 ± 10.3	1.26 ± 0.38	1.59 ± 0.48*	0.22 ± 0.07**
8	DmTrxR (Δ8)	24.6 ± 5.03	2.69 ± 0.57**	2.12 ± 0.44**	0.08 ± 0.54**
9	PfTrxR (GCGGGKUG)	751 ± 173	1.33 ± 0.39	-	0.16 ± 0.17**
10	PfTrxR (GCGGGKCG)	632 ± 14.0	1.10 ± 0.22	-	1.54 ± 0.12**
11	PfTrxR (GCGGGKSG)	38.7 ± 5.39	1.75 ± 0.25**	-	1.55 ± 0.24**
12	PfTrxR (GCUG)	93.9 ± 18.7	2.29 ± 0.49**	-	0.06 ± 0.01**
13	PfTrxR (GCCG)	232 ± 35.9	1.25 ± 0.27	-	2.19 ± 0.73**
14	PfTrxR (Δ7)	198 ± 8.66	0.93 ± 0.14	-	2.54 ± 0.31**

The number of asterisk indicate significant differences compared to the control

^*p <0.05,

^**p <0.01).

These results are displayed in Fig. 5 as fold change in V₀ with enzymes grouped into Sec-containing and non-Sec-containing enzymes. Four of the five Sec-containing enzymes were inhibited by Man-A, with the single exception being mTrxR-2 (GCUG). Of the eight non-Sec-containing TrxRs, six were activated by Man-A. The two which were inhibited were DmTrxR (SCCS) and DmTrxR (Δ8). Among the truncated enzymes, mTrxR-2 (Δ8) and PfTrxR (Δ7) were activated and only DmTrxR (Δ8) was inhibited. Interestingly, all DmTrxR enzymes were inhibited by Man-A regardless of the composition of the C-terminal redox center.

Fig 5.

Fold change in the initial rate of DTNB reduction by TrxR in the presence of Man-A (20 μM). Solid black bars are Sec-containing enzymes. White bars are non-Sec-containing enzymes.

3.3. Test for reversibility of activation of DTNB reduction by rTrxR-1 and mTrxR-2.

In order to determine if the inhibition or activation of TrxR by Man-A or PbTx-2 is reversible, the DTNB reduction assay was performed as before however, after a 1 hour incubation of pre-reduced TrxR with either PbTx-2 or Man-A, the mixture was passed through a gel filtration column (MW cutoff of 6000 amu) followed by a 30 min incubation with additional NADPH (Fig. 6). Gel filtration should remove all unbound compound from the solution and the subsequent 30 min incubation should allow for reestablishing equilibrium. The normal activity of rTrxR-1 is restored after passage of the rTrxR-1/PbTx-2 mixture through a size exclusion column (Fig. 6A). Similarly, the normal activities of ManA-treated mTrxR-2 enzymes (GCUG, GCCG and GSSG) are restored by gel filtration (Fig. 6B-D). On the other hand, the activities of Man-A treated rTrxR-1, DmTrxR (SCUG), DmTrxR (SCCS), DmTrxR (Δ8), PfTrxR (GCUG) and PfTrxR (GKUG) are not recovered after gel filtration (Fig 6A, E-H). (Tuladhar and Rein, 2018).

Fig. 6.

Reduction of DTNB (2 mM) by TrxR (5.2 nM) after gel filtration of TrxR/effector mixture. (A) rTrxR-1. (B) mTrxR-2 (GCUG). (C) mTrxR-2 (GCCG). (D) mTrxR-2 (GSSG); (E) DmTrxR (SCUG); (F) DmTrxR (Δ8) (G) DmTrxR (SCCS) (H) PfTrxR (GKUG) (I) PfTrxR (GCUG) PbTx-2 (Δ, 20 μM), Man-A (□, 2 μM for rTrxR-1 and 4.2 μM for TrxR-2 and mutants and 20 μM for Dm and Pf enzymes), Control (DMSO, ○). Man-A data from panel A was adapted with permission from (Tuladhar and Rein, 2018).

3.4. Reduction of H₂O₂.

The reduction of H₂O₂ by TrxR requires a fully functional C-terminal redox center with an uncompromised redox active Sec residue (Zhong and Holmgren, 2000). The hydrogen peroxidase activity of Sec-containing TrxR enzymes was evaluated after 15 minutes of incubation of the reduced TrxR with Man-A or PbTx-2 (Table 3). Man-A inhibited all Sec-containing TrxR enzymes. Interestingly, mTrxR-2 (GCUG) was only modestly inhibited in the presence of Man-A with 75% of the original activity retained. In the presence of PbTx-2 only two enzymes were different from the control: rTrxR-1 (GCUG) was slightly inhibited by 0.74-fold relative to the control and mTrxR-2 (GCUG) which was activated by 1.4-fold relative to the control.

Table 3.

Rates of H₂O₂ reduction by Sec-containing TrxR enzymes after 15 minute incubation with Man-A or PbTx-2 (38.5 μM). Data expressed as NADPH consumed (mol/min-mol enzyme).

Enzyme		V0 Control Rate	Fold change in V0 relative to control
			PbTx-2	Man-A
1	rTrxR-1 (GCUG)	185 ± 21.0	0.74 ± 0.10*	0.07 ± 0.08 **
2	mTrxR-2 (GCUG)	235 ± 22.6	I.44 ± 0.14**	0.75 ± 0.10 *
6	DmTrxR (SCUG)	65.5 ± 16.5	0.78 ± 0.34	No activity **
9	PfTrxR (GCGGGKUG)	116 ± 8.8	0.90 ± 0.34	0.31 ± 0.10**
10	PfTrxR (GCUG)	66.6 ± 17.8	0.89 ± 0.25	0.03 ± 0.10 **

The number of asterisk indicate significant differences compared with control

^*p <0.05,

^**p <0.01)

3.5. Insulin reduction

The rate of Trx reduction was monitored using an insulin reduction assay (Montano et al., 2014). In this two-enzyme assay, oxidized Trx is continuously reduced by TrxR with reducing equivalents ultimately provided by NADPH. The fluorescent signal is only possible when insulin is reduced by Trx. The effect of Man-A and PbTx-2 (20 μM) on Trx reduction by rTrxR-1 and mTrxR-2 (CGUG) and of Man-A on DmTRxR (SCUG), PfTrxR (GCUG) was examined in this assay (Fig. 7). The activities of other enzymes were undetectable in this assay. Both Man-A and PbTx-2 are inhibitors of Trx reduction by rTrxR-1 (Fig. 7A) with IC₅₀ values of 572 nM and 25 μM respectively (Chen et al., 2017; Tuladhar and Rein, 2018). As shown in Fig. 7B, PbTx-2 has no effect on Trx reduction by mTrxR-2 (GCUG) whereas Man-A completely inhibits Trx reduction by all TrxR enzymes at 20 μM (Fig. 7A-D). However, from this two enzyme assay it is impossible to determine if TrxR or Trx is being inhibited.

Fig.7.

Inhibition of insulin reduction by (A) rTrxR-1 (B) mTrxR2 (CGUG) (100 nM); (C) DmTRxR (SCUG) (200 nM); (D) PfTrxR (GCUG) (200 nM) in the presence of Man-A (20 μM, □); PbTx-2 (20 μM, ◆); PbTx-3 (20 μM, ∎); Control (∘). Data from panel A was adapted with permission from (Chen et al., 2017; Tuladhar and Rein, 2018).

3.6. Modified papain assay

In order to determine if Man-A inhibits Trx, the modified papain assay (Singh et al., 1995) was performed. This assay is based on the activation of inactive papain methylsulfide by Trx. Activated papain then reduces its substrate and the rate is dependent on the amount of activated papain, which is in turn dependent on Trx. We first examined the effect of Man-A at 20 μM on papain and determined that papain is not inhibited (data not shown). We next determined that the activation of papain methylsulfide by Trx is not affected by Man-A at 20 μM. We may therefore conclude that Man-A inhibits Trx reduction by all TrxR enzymes tested. This experiment would not be informative for PbTx-2 because it is complicated by the fact that PbTx-2 is an inhibitor of papain at concentrations which are comparable to the IC₅₀ for inhibition of insulin reduction by rTrxR-1 (Sudarsanam et al., 1992). However, the lack of inhibition by PbTx-2 in the insulin reduction assay with mTrxR-2 (GCUG) confirms that PbTx-2 does not inhibit Trx.

3.7. Reduction of selenocystine.

The rate of reduction of the diselenide, selenocystine was evaluated for TrxR enzymes after 15 minutes of incubation with PbTx-2 or Man-A as shown in Table 4. The C-terminal Sec is believed to be required for the efficient reduction of selenocystine. This is supported by the lower activity seen in the mTrxR-2 (GCCG) when compared to the wild type mTrxR-2 (GCUG), DmTrxR (SCCS) vs DmTrxR (SCUS), PfTrxR (GCCG) vs PfTrxR (GCUG) and PfTrxR (GKCG) vs PfTrxR (GKUG). PbTx-2 either activates (mTrxR-2 (GCUG, GCCG, Δ8) and DmTrxR (Δ8)) or does not affect the rate of selenocystine reduction by TrxR. Man-A inhibits selenocystine reduction in all Sec-containing enzymes with the exception of mTrxR-2 (GCUG). Also inhibited were mTrxR-2 (GCCG), DmTrxR (SCCS) and PfTrxR (GCCG). No change was observed for mTrxR-2 (GSSG), PfTrxR (GKCG) and PfTrxR (GKSG). mTrxR-2 (GCUG) was activated as were all truncated enzymes: mTrxR-2 (Δ8), DmTrxR (Δ8) and PfTrxR (Δ7).

Table 4.

Rates of selenocystine (120 μM) reduction by mammalian TrxR enzymes after 15 min incubation with Man-A or PbTx-2 (38.5 μM). Data expressed as NADPH consumed (mol/min-mol enzyme).

Enzyme		V0 Control Rate	Fold change in V0 relative to control
			PbTx-2	Man-A
1	rTrxR-1 (GCUG)	337 ± 80.5	1.21 ± 0.3	0.04 ± 0.19**
2	mTrxR-2 (GCUG)	347 ± 21.5	1.25 ± 0.10**	1.59 ± 0.32*
3	mTrxR-2 (GCCG)	82.3 ± 18.8	1.60 ± 0.41**	No activity*
4	mTrxR-2 (GSSG)	No activity	[11.1 ± 5.58]† **	No activity
5	mTrxR-2 (Δ8)	No activity	[33.1 ± 18.9]† **	[39.16 ± 21.6]†*
6	DmTrxR (SCUG)	976 ± 127	1.09 ± 0.16	0.04 ± 0.04**
7	DmTrxR (SCCS)	148 ± 15.2	1.23 ± 0.13	0.25 ± 0.16**
8	DmTrxR (Δ8)	No activity	[37.5 ± 15.32]†	[51.6 ± 26.0]†
9	PfTrxR (GCGGGKUG)	182 ± 43.6	1.12 ± 0.32	0.05 ± 0.11**
10	PfTrxR (GCGGGKCG)	15.9 ± 7.91	1.16 ± 0.67	No activity**
11	PfTrxR (GCGGGKSG)	No activity	No activity	No activity
12	PfTrxR (GCUG)	123 ± 53.6	1.50 ± 0.69	No activity**
13	PfTrxR (GCCG)	34.1 ± 7.5	1.31 ± 0.32	No activity**
14	PfTrxR (Δ7)	36.4 ± 9.4	1.30 ± 0.35	1.47 ± 0.42

The number of asterisk indicate significant differences compared with control

^*p <0.05,

^**p <0.01)

^†actual rate.

3.8. NADPH oxidase activity.

The consumption of NADPH by TrxR enzymes in the presence of Man-A or PbTx-2 (38.5 μM) and in the absence of other substrates was monitored (Table 5). PbTx-2 activated TrxR prooxidant activity in three enzymes: mTrxR-2 (GSSG and Δ8) as well as PfTrxR (GSKG). NADPH oxidase activity was not significantly different from the control rates for all other enzymes. Man-A activated NADPH oxidation in all enzymes except for three Pf enzymes (GKSG, GCCG and 7).

Table 5.

NADPH oxidase activity in mammalian TrxR enzymes in the presence of Man-A or PbTx-2/3 (38.5 μM). Data expressed as NADPH consumed (mol/min-mol enzyme)

Enzyme		V0 Control Rate	Fold change in V0 relative to control
			PbTx-2	Man-A
1	rTrxR-1 (GCUG)	1.13 ± 0.09	2.51 ± 1.46	15.53 ± 2.14**†
2	mTrxR-2 (GCUG)	0.93 ± 0.14	1.26 ± 1.31	4.04 ± 1.70**
3	mTrxR-2 (GCCG)	0.40 ± 0.15	4.13 ± 3.46	3.45 ± 1.36**
4	mTrxR-2 (GSSG)	0.11 ± 0.04	25.45 ± 9.38**	13.91 ± 5.14**
5	mTrxR-2 (Δ8)	0.49 ± 0.43	2.41 ± 2.28*	3.88 ± 4.08**
6	DmTrxR (SCUG)	3.19 ± 1.01	0.95 ± 0.57	3.04 ± 1.12**
7	DmTrxR (SCCS)	0.58 ± 0.30	1.76 ± 1.33	16.1 ± 10.8*
8	DmTrxR (Δ8)	1.10 ± 0.01	1.70 ± 0.90	13.4 ± 5.35*
9	PfTrxR (GCGGGKUG)	7.96 ± 0.87	1.11 ± 0.58	1.60 ± 0.20**
10	PfTrxR (GCGGGKCG)	2.05 ± 0.004	1.42 ± 1.07	2.39 ± 0.51*
11	PfTrxR (GCGGGKSG)	3.91 ± 0.66	1.45 ± 0.16*	1.45 ± 0.26
12	PfTrxR (GCUG)	1.08 ± 0.63	1.75 ± 1.32	4.00 ± 2.49**
13	PfTrxR (GCCG)	2.82 ± 0.22	1.32 ± 0.31	2.38 ± 1.46
14	PfTrxR (Δ7)	18.83 ± 3.96	1.00 ± 0.18	1.45 ± 0.45

The number of asterisk indicate significant differences compared with control

^*p <0.05,

^**p <0.01).

^†62.5 μM Man-A

3.9. DTNB reduction competition assay.

The rTrxR-1 inhibitor curcumin is known to react with the C-terminal Sec residue of rTrxR-1. We have previously shown that pre-incubation of PbTx-2 with rTrxR-1 prevents inhibition by curcumin (Chen et al., 2017) suggesting that these two inhibitors act at the same site. Manumycin and PbTx-2 are both believed to act at the C-terminal redox center of rTrxR-1, but with very different outcomes. Man-A is an inhibitor and PbTx-2 is an activator of DTNB reduction. Fig. 8 shows the dose response curves for DTNB reduction by rTrxR-1 in the presence of increasing concentrations of Man-A with or without pre-incubation with PbTx-2 (20 μM, 30 min). While the dose response curve is steeper in the presence of PbTx-2, the IC₅₀ for Man-A does not change significantly; 1586 (± 128) nM for Man-A alone vs 1795 (± 208) nM for Man-A in the presence of 20 μM PbTx-2.

Fig. 8.

Inhibition of DTNB (2 mM) reduction by rTrxR-1 (5.6 nM) in presence of Man-A (0-5000 nM) and PbTx-2 (20 μM). Preincubated with brevetoxin for 30 min prior to Man-A (□) and no PbTx-2 added (×).

4. Discussion

Using a series of enzymatic assays, the behavior of two TrxR effectors, PbTx-2 and Man-A, was evaluated with a suite of TrxR enzymes from a variety of sources, including mammalian, insect and protist, as well as several mutants. Each assay provides unique information on the nature of the interaction between the enzyme and effector.

In addition to Trx, TrxR can reduce other targets such as the non-native disulfide DTNB, the diselenide selenocystine and H₂O₂. Under most circumstances, the reduction of these small molecule substrates likely occurs via the C-terminal redox center and the rate of reduction of these substrates is typically accelerated by the presence of Sec. Zhong and Holmgren reported that the TrxR-1 Sec to Cys (GCCG) mutant reduces DTNB at a rate of only 5% of the control (Zhong and Holmgren, 2000). Although less pronounced, this trend holds for the mTrxR-2 series (GCUG vs. GCCG), the DmTrxR (SCUG vs. SCCS) series and the PfTrxR series (GKUG vs. GKCG) but not for PfTrxR (GCUG vs. GCCG). Under some circumstances, DTNB reduction may be catalyzed by the N-terminal redox center (Cheng et al., 2009; Lothrop et al., 2009). The TrxR enzymes having a “dead tail”, mTrxR-2 (GSSG) and PfTrxR (GKSG) or a truncated tail mTrxR-2 (Δ8), DmTrxR (Δ8) and PfTrxR (Δ7) must rely solely on the N-terminal redox center. It has been reported that the rate determining step in the flow of reducing equivalents is the reduction of the C-terminal redox center by the N-terminal redox center (Snider et al., 2014). It is perhaps noteworthy that, among the mTrxR-2 enzymes, the untreated mTrxR-2 (GSSG) or “dead tail” mutant had the slowest rate of DTNB reduction and the untreated mTrxR-2 (Δ8) had the fastest rate. From this, one must conclude that the reduction of small substrates like DTNB is faster when the N-terminal redox center is unobstructed and the electron flow can bypass the C-terminal redox center.

Selenocystine reduction occurs principally at the C-terminal redox center. Like DTNB reduction, the rate is accelerated by the presence of Sec. This effect is illustrated by the following pairs of enzymes: mTrxR-2 (GCUG vs. GCCG), DmTrxR (SCUG vs. SCCS), PfTrxR (GKUG vs. GKCG) and PfTrxR (GCUG vs. GCCG). Among the truncated enzymes, only PfTrxR (Δ7) is able to reduce selenocystine via the N-terminal redox center. On the other hand, the reduction of H₂O₂ has an absolute requirement for a fully functional C-terminal redox center with an uncompromised Sec residue (Zhong and Holmgren, 2000). Therefore, only Sec-containing enzymes were tested for this activity. The reduction of Trx by TrxR is preceded by docking of the two enzymes through a series of hydrophobic and electrostatic interactions(Fritz-Wolf et al., 2011). Once docked, the C-terminal arm of TrxR rotates towards Trx. The reduction of Trx by TrxR requires a fully functional redox center and an unobstructed interface between the two enzymes. Finally, under normal circumstances, the NADPH oxidase activity of TrxR enzymes is low (Table 5). When the C-terminal redox center is inactivated, TrxR can become a SecTRAP (selenium compromised thioredoxin reductase-derived apoptotic protein). This can occur via alkylation of the Sec residue with electrophiles (Gan et al., 2013) or coordination with some noble metals such as gold or platinum (Anestål et al., 2008). SecTRAPs use NADPH to produce O₂^−• via the N-terminal redox center.

Man-A is a more potent effector than PbTx-2/-3 and Man-A behaves like a typical inhibitor towards most TrxR enzymes. At less than 4 nM Man-A is able to block activation of DTNB reduction by rTrxR-1 in the presence of 20 μM PbTx-2. Man-A inhibits DTNB reduction for all but one (mTrxR-2) Sec-containing TrxR whereas PbTx-2/-3 activate DTNB reduction for most enzymes and does not discriminate between Sec-containing and non-Sec-containing enzymes. Both PbTx-2 and Man-A are able to activate NADPH oxidase activity even in the absence of covalent modification of Sec, although the differences for PbTx-2 are not significant.

4.1. Effect of brevetoxins on the activity of various TrxRs.

Table 6 summarizes the effects of all enzymes which had activated DTNB reductase activity in the presence of PbTx-2. Those enzymes whose DTNB activity was unaffected by PbTx-2 were also unaffected in all other assays. PbTx-2 activates DTNB reduction by all mammalian TrxR and two out of three of the Drosophila enzymes (SCUG and Δ8) as well as two Plasmodium enzymes, the chimera PfTrxR (GCUG) and the “dead tail” P. falciparum mutant enzyme PfTrxR (GKSG). A modest, but not statistically significant activation was observed for DmTrxR (SCCS). The previously proposed mechanism of TrxR-1 activation by PbTx-2 entailed the formation of a Michael adduct between Sec498 and the α,β-unsaturated aldehyde on the K-ring side chain of the brevetoxin molecule, inhibiting Trx reduction by the C-terminal redox center (Chen et al., 2017). It was further proposed that the large size of the brevetoxin molecule prevents the C-terminal extension from folding back towards the N-terminal disulfide. The normally inaccessible N-terminal redox center therefore becomes accessible to small molecule substrates. However, even though PbTx-3 (and by analogy PbTx-2) react readily with Sec, subsequent experiments suggest that a stable covalent adduct is not formed between the Sec-containing TrxR enzymes and PbTx-2 or -3. When an rTrxR-1/PbTx-2 mixture is passed through a size exclusion column, followed by a 30 minute equilibration, normal activity with respect to DTNB reduction is restored. This would suggest that the interaction between PbTx-2 and rTrxR-1 is non-covalent or reversible. The activation of the “dead tail” enzymes as well as the truncated mutants demonstrates that covalent modification of the Sec residue is not a prerequisite for DTNB activation. Indeed, the mTrxR-2 (GSSG) or “dead tail” mutant experienced the greatest degree of activation by PbTx-2 and PbTx-3 (9-10 fold). The rates of DTNB reduction for the mTrxR-2 (GSSG) and (Δ8) in the presence of PbTx-2 or PbTx-3 were among the highest measured and were statistically indistinguishable from each other. We propose that the brevetoxins associates with mammalian TrxR enzymes in such a way that the enzymes undergoes a conformational change exposing the N-terminal redox center resulting in the maximum possible rate of DTNB reduction. As previously reported (Chen et al., 2017) PbTx-2 only modestly inhibits Trx reduction (IC₅₀ = 25 μM) by rTrxR-1, but did not affect Trx reduction by TrxR-2. PbTx-2 slightly inhibited hydrogen peroxidase activity of rTrxR-1, enhanced this activity in mTrxR-2 and had no effect on the other Sec-containing enzymes. Both Trx and H₂O₂ reduction require a functional C-terminal redox center and hydrogen peroxidase activity requires an unmodified C-terminal Sec (Zhong and Holmgren, 2000). Furthermore, the reduction of selenocystine by TrxR enzymes is either unaffected or activated by PbTx-2. Indeed, three enzymes (mTrxR-2 (GSSG), mTrxR-2 (Δ8) and DmTrxR (Δ8)) none of which have a functional C-terminal redox center and whose selenocystine reductase activity is undetectable in the absence of an effector, were activated in the presence of PbTx-2. This observation again, supports the exposure of the N-terminal redox center but not adduction of the C-terminal redox center. The modest inhibition of Trx and hydrogen peroxide reduction by rTrxR-1 in the presence of PbTx-2 may be the result of the reversible formation of a thermodynamically disfavored covalent adduct between PbTx-2 and the C-terminal Sec of rTrxR-1. This notion is supported by the fact that PbTx-3, which lacks the α, β-unsaturated carbonyl showed no inhibition of Trx reduction (Chen et al., 2017) even though it did activate DTNB reduction by all enzymes tested (Table 2, entries 1-8). Finally Man-A, which does form a stable adduct with rTrxR-1 (Tuladhar and Rein, 2018) is able to inhibit DTNB reduction by rTrxR-1 even in the presence of PbTx-2.

Table 6:

The effect of PbTx-2 on various activities of thioredoxin reductase and the rational for the observed effect.

DTNB activity	hydrogen peroxidase activity	selenocystine reductase activity	NADPH oxidase activity
↑ rTrxR-1 (GCUG)§ mTrxR-2 (GCUG) mTrxR-2 (GCCG) mTrxR-2 (GSSG) mTrxR-2 (Δ8) DmTrxR (SCUG) DmTrxR (Δ8) PfTrxR (GKSG) PfTrxR (GCUG)	↓ rTrxR-1 (GCUG) ↑ mTrxR-2 (GCUG) does not apply to others	↑ mTrxR-2 (GCUG) mTrxR-2 (GCCG) mTrxR-2 (GSSG) mTrxR-2 (Δ8) DmTrxR (Δ8) Unchanged rTrxR-1 (GCUG) DmTrxR (SCUG) PfTrxR (GKSG) PfTrxR (GCUG)	↑↑↑ mTrxR-2 (GSSG) ↑ mTrxR-2 (Δ8) PfTrxR (GKSG) increased but not significant rTrxR-1 (GCUG) mTrxR-2 (GCUG) mTrxR-2 (GCCG) DmTrxR (SCUG) DmTrxR (Δ8) Unchanged PfTrxR (GCUG)
DTNB-reductase activity in TrxR1 depends upon an unmodified selenol. Since the activity increased, PbTx-2 must not form an adduct. Non-covalent binding of PbTx-2 results in a conformational change causing the N-terminal redox center to become exposed, resulting in an increase in activity. §Normal activity is recovered after gel filtration. Others were not tested with PbTx-2	Hydrogen peroxidase activity is very strongly dependent upon an unmodified selenol. The increase in activity is consistent with the absence of adduct formation (mTrxR-2). The modest decrease (mTrxR-1) may be due to obstruction of the active site or incomplete formation of an unstable adduct.	Selenocystine-reductase activity is accelerated by an unmodified selenol. The unchanged or modest increase in activity is consistent with the absence of adduct formation.	The increase in NADPH oxidase activity is consistent with non-covalent binding instead of adduct formation. Non-covalent binding of PbTx-2 results in a conformational change causing the N-terminal redox center to become exposed, resulting in an increase in activity. Only enzymes with a non-functional C-terminal redox center are activated.

The chemical modification of the C-terminal Sec of TrxR-1 yields a SecTRAP (selenium compromised thioredoxin reductase-derived apoptotic protein) promoting both apoptosis and necrosis via oxidative stress and increased intracellular reactive oxygen species (ROS) production (Anestål et al., 2008). Both curcumin and juglone modified TrxR-1 have demonstrated strongly induced NADPH oxidase activity, producing O₂^−• in the presence of oxygen via the N-terminal (Cys59/Cys64) redox center (Cenas et al., 2004; Fang and Holmgren, 2006; Powis et al., 2006). Most TrxR enzymes experienced and increase in NADPH consumption when treated with PbTx-2. However, this increase was significant only for the mTrxR-2 (GSSG) and the PfTrxR (GKSG) “dead tail” mutants. Interestingly, mTrxR-2 (GSSG) also experienced the largest enhancement of DTNB reduction. This observation demonstrates that chemical modification of the C-terminal redox center is not required for the formation of a SecTRAP.

4.2. Effect of Man-A on the activity of various TrxRs.

The behavior of Man-A towards TrxR enzymes is more complex and is summarized in Table 7. The rate of DTNB reduction by all of the TrxR enzymes tested is affected in some way by Man-A. In some instances, the reaction is inhibited and in others it is accelerated. With the exception of mTrxR-2 (GCUG), the reduction of DTNB by Sec-containing enzymes was inhibited by Man-A. With the exception of DmTrxR (SCCS) and DmTrxR (Δ8), the reduction of DTNB by non Sec-containing enzymes was activated by Man-A. Size exclusion chromatography of Sec-containing-TrxR/Man-A solutions failed to restore activity of rTrxR-1, DmTrxR (SCUG), DmTrxR (SCCS), DmTrxR (Δ8), PfTrxR (GCUG) and PfTrxR (GKUG), but did restore normal activity for mTrxR-2 (Fig.6B). Man-A inhibited H₂O₂ reduction by all Sec-containing enzymes. However, the inhibition of mTrxR-2 (GCUG) was modest with 75% of the activity remaining. The effect of Man-A on selenocystine reduction was variable: seven enzymes were inhibited, three enzymes were activated and three enzymes were not statistically different from the control. Among the enzymes which were activated were four of the five Sec-containing enzymes with the exception of mTrxR-2 (GCUG) which experienced a 1.6-fold activation. Other inhibited enzymes were m-TrxR-2 (GCCG), DmTrxR (SCCS) and PfTrxR (GCCG). Other enzymes which were activated were the truncated enzymes: m-TrxR-2 (Δ8), DmTrxR (Δ8). PfTrxR (Δ7) experienced a 1.47-fold activation, but was not statistically different from the control. Two of these truncated enzymes (m-TrxR-2 (Δ8) and DmTrxR (Δ8)) had no detectable selenocystine reductase activity in the absence of effector. Enzymes which were unaffected were m-TrxR-2 (GSSG), PfTrxR (GKCG) and PfTrxR (GKSG). These data indicate that Man-A forms a covalent adduct with the Sec residue of Sec-containing TrxR enzymes with the single exception of mTrxR-2. Surprisingly, after gel filtration of a solution of Man-A and DmTrxR (SCCS) or DmTrxR (Δ8), the V₀ for DTNB reduction was restored to only 36% and 60% of the original activity. This indicates that under some circumstances, Man-A can react with Cys, but more importantly, that Man-A can also react with the Cys residues of the N-terminal redox center. Given that mTrxR-2 is not adducted, it was somewhat surprising that Man-A completely inhibited the reduction of Trx by mTrxR-2. We therefore established that Trx is not inhibited by Man-A using a modified papain assay. It seems curious that the reduction of Trx by mTrxR-2 (GCUG) is completely inhibited at 20 μM whereas the reduction of H₂O₂ is only reduced by 25% at 38.5 μM. Man-A behaves towards TrxR-2 (GCUG) very much like PbTx-2 behaves towards rTrxR-1: activation of DTNB reduction, slight inhibition of H₂O₂ reduction and inhibition of Trx reduction. All experimental data would indicate that a stable covalent adduct is not formed between mTrxR-2 (GCUG) and Man-A. The inhibition of Trx reduction may be a matter of the relative sizes of the substrates. Trx is a large substrate relative to H₂O₂ and requires initial docking to TrxR prior to reduction (Fritz-Wolf et al., 2011). It is possible that Man-A disrupts the electrostatic and hydrophobic docking interactions between Trx and TrxR even in the non-covalent complex. This may also be the case for PbTx-2 and rTrxR-1. Finally, NADPH oxidase activity is induced by Man-A in all TrxR enzymes including those that do not form a covalent adduct such as the “dead tail” and truncated mutants of mTrxR-2 and PfTrxR. In fact, mTrxR-2 (GCUG) and mTrxR-2 (GSSG) were among the most highly activated pro-oxidants. As we saw with PbTx-2, this observation confirms that covalent modification of the Sec residue is not required to induce TrxR pro-oxidant activity.

Table 7:

The effect of Man-A on various activities of thioredoxin reductase and the rational for the observed effect.

DTNB activity	hydrogen peroxidase activity	selenocystine reductase activity	NADPH oxidase activity
↓ rTrxR-1 (GCUG) DmTrxR (SCUG) DmTrxR (SCCS) DmTrxR (Δ8) PfTrxR (GKUG) PfTrxR (GCUG)	↓↓ rTrxR-1 (GCUG) DmTrxR (SCUG) PfTrxR (GKUG) PfTrxR (GCUG) does not apply to; DmTrxR (SCCS) DmTrxR (Δ8)	↓↓ rTrxR-1 (GCUG) DmTrxR (SCUG) DmTrxR (SCCS) PfTrxR (GKUG) PfTrxR (GCUG) ↑ DmTrxR (Δ8)	↑↑ rTrxR-1 (GCUG) DmTrxR (SCCS) DmTrxR (Δ8) ↑ DmTrxR (SCUG) PfTrxR (GKUG) PfTrxR (GCUG)
DTNB-reductase activity in TrxR1 depends upon an unmodified selenol and accelerates the rate for other enzymes. Normal activity is not recovered after gel filtration. Data is consistent with formation of a Sec-Man-A adduct or Cys-Man-A adduct for DmTrxR (Δ8) and (SCCS). Partial activity is recovered for DmTrxR (Δ8) indicates that alkylation of Cys-by Man-A is not quantitative.	Hydrogen peroxidase activity is very strongly dependent upon an unmodified selenol. The large decrease in activity is consistent with formation of a Sec-Man-A adduct.	Selenocystine-reductase activity is accelerated by an unmodified selenol. The large decrease in activity is consistent with formation of a Sec-Man-A adduct. DmTrxR (Δ8) acquires activity by exposure of the N-terminal redox center.	The very large increase in NADPH oxidase activity is consistent with formation of a SecTRAP. The modified enzyme undergoes a conformational change that results in a large increase in oxidase activity.
↑	↓	↑↑	↑↑
mTrxR-2 (GCUG)§ mTrxR-2 (GCCG)§ mTrxR-2 (GSSG)§ mTrxR-2 (Δ8) PfTrxR (GKCG) PfTrxR (GKSG) PfTrxR (GCCG) PfTrxR (Δ7) §Normal activity is recovered after gel filtration. Others enzymes were not tested	mTrxR-2 (GCUG)	mTrxR-2 (Δ8)	mTrxR-2 (GSSG)
		↑	↑
	(does not apply to others)	mTrxR-2 (GCUG)	mTrxR-2 (GCUG) mTrxR-2 (GCCG) mTrxR-2 (Δ8) PfTrxR (GKCG)
		↓↓
		mTrxR-2 (GCCG) PfTrxR (GCCG) PfTrxR (GKCG)
			Unchanged
		Unchanged	PfTrxR (GKSG) PfTrxR (GCCG) PfTrxR (Δ7)
		mTrxR-2 (GSSG) PfTrxR (Δ7) PfTrxR (GKSG)
Binding of ManA to TrxR1 results in a conformational change that results in the N-terminal redox center being more available, resulting in an increase in activity.	The slight decrease in activity is consistent with noncovalent binding instead of adduct formation.	The increase in activity is consistent with noncovalent binding instead of adduct formation.	The small increase in NADPH oxidase activity is consistent with noncovalent binding instead of adduct formation.

In summary, we have demonstrated that brevetoxins and Man-A influence the functionality of TrxR enzymes. The outcome is dependent on the combination of effector and specific ortholog of TrxR. We envision three possible pathways for altering the activity of TrxR: an initial non-covalent binding event-which (i) induces a conformational change to the enzyme exposing the N-terminal redox or (ii) blocks the approach of substrates or (iii) is followed by the formation of a covalent adduct with the enzyme depending on the combination of enzyme and effector. All three compounds are capable of reacting with Sec. Nonetheless, PbTx-2/3 do not appear to form an adduct with any of the enzymes with the possible exception of an unstable adduct formed between PbTx-2 and rTrxR-1, based on modest inhibition of hydrogen peroxidase activity and Trx reduction. A covalent adduct is likely formed between Sec-containing enzymes and Man-A with the exception of TrxR-2 (GCUG). The most likely site of adduct formation is the C-terminal Sec, as DTNB reduction is activated for most non-Sec-containing enzymes and reaction at the N-terminal redox center would inhibit DTNB reduction. One non-Sec enzyme DmTrxR (SCCS) must react at a Cys residue and one truncated enzyme DmTrxR (Δ8) must react at the N-terminal redox center. For the Sec-containing enzymes, we can’t rule out reaction at both redox centers. The formation of a covalent adduct at the Sec residue of TrxRs is known to induce NADPH oxidase activity. However, we have shown that covalent adduct formation is not a prerequisite for this gain of function as Man-A induces NADPH oxidase in all enzymes regardless of the nature of the interaction. This work also underscores the significant differences between the two mammalian orthologs TrxR-1 and TrxR-2.

Highlights.

• An alternate mechanism of brevetoxin toxicity has been identified.

• Covalent modification of TrxR is not required for NADPH oxidase activity.

• Manumycin-A can discriminate between cytosolic and mitochondrial TrxR.