Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA.

Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 ß subunit (eIF2ß). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.

The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues.

The brevetoxins, neurotoxins produced by Karenia brevis, the Florida red tide dinoflagellate, effect fish and wildlife mortalities and adverse public health and economic impacts during recurrent blooms. Knowledge of the biochemical consequences of toxin production for K. brevis could provide insights into an endogenous role of the toxins, yet this aspect has not been thoroughly explored. In addition to neurotoxicity, the most abundant of the brevetoxins, PbTx-2, inhibits mammalian thioredoxin reductase (TrxR). The thioredoxin system, composed of the enzymes TrxR and thioredoxin (Trx), is present in all living organisms and is responsible in part for maintaining cellular redox homeostasis. Herein, we describe the cloning, expression, and semisynthesis of the selenoprotein TrxR from K. brevis (KbTrxR) and reductase activity toward a variety of substrates. Unlike mammalian TrxR, KbTrxR reduces oxidized glutathione (GSSG). We further demonstrate that PbTx-2 is an inhibitor of KbTrxR. Covalent adducts between KbTrxR and rat TrxR were detected by mass spectrometry. While both enzymes are adducted at or near the catalytic centers, the specific residues are distinct. Biochemical differences reported for high and low toxin producing strains of K. brevis are consistent with the inhibition of KbTrxR and suggest that PbTx-2 is an endogenous regulator of this critical enzyme.

Essential components of the xanthophyll cycle differ in high and low toxin Karenia brevis.

The dinoflagellate Karenia brevis, blooms annually in the Gulf of Mexico, producing a suite of neurotoxins known as the brevetoxins. The cellular toxin content of K. brevis, however, is highly variable between or even within strains. Herein, we investigate physiological differences between high (KbHT) and low (KbLT) toxin producing cultures both derived from the Wilson strain, related to energy-dependent quenching (qE) by photosystem II, and reduced thiol content of the proteome. We demonstrate that gene and protein expression of the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (Dde) and monogalactosyldiacylglycerol (MGDG) synthase are not significantly different in the two cultures. Using redox proteomics, we report a significantly higher reduced cysteine content in the low toxin proteome, including plastid localized thioredoxin reductase (Trx) which can result in inactivation of Dde and activation of MGDG synthase. We also report significant differences in the lipidomes of KbHT and KbLT with respect to MGDG, which facilitates the xanthophyll cycle.

Effectors of thioredoxin reductase: Brevetoxins and manumycin-A.

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.

Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O2-•).

Brevetoxin (PbTx-2) influences the redox status and NPQ of Karenia brevis by way of thioredoxin reductase.

The Florida red tide dinoflagellate, Karenia brevis, is the major harmful algal bloom dinoflagellate of the Gulf of Mexico and plays a destructive role in the region. Blooms of K. brevis can produce brevetoxins: ladder-shaped polyether (LSP) compounds, which can lead to adverse human health effects, such as reduced respiratory function through inhalation exposure, or neurotoxic shellfish poisoning through consumption of contaminated shellfish. The endogenous role of the brevetoxins remains uncertain. Recent work has shown that some forms of NADPH dependent thioredoxin reductase (NTR) are inhibited by brevetoxin-2 (PbTx-2). The study presented herein reveals that high toxin and low toxin K. brevis, which have a ten-fold difference in toxin content, also show a significant difference in their ability, not only to produce brevetoxin, but also in their cellular redox status and distribution of xanthophyll cycle pigments. These differences are likely due to the inhibition of NTR by brevetoxin. The work could shed light on the physiological role that brevetoxin fills for K. brevis.

Toxin composition of the 2016 Microcystis aeruginosa bloom in the St. Lucie Estuary, Florida.

A bloom of the cyanobacteria, Microcystis aeruginosa occurred in the St. Lucie Estuary during the summer of 2016, stimulated by the release of waters from Lake Okeechobee. This cyanobacterium produces the microcystins, a suite of heptapeptide hepatotoxins. The toxin composition of the bloom was analyzed and was compared to an archived bloom sample from 2005. Microcystin-LR was the most abundant toxin with lesser amounts of microcystin variants. Nodularin, cylindrospermopsin and anatoxin-a were not detected.

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

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, ß-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, ß-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.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light-Harvesting Complex II (LHCII) of Photosystem II.

The brevetoxins are neurotoxins that are produced by the "Florida red tide" dinoflagellate Karenia brevis. They bind to and activate the voltage-gated sodium channels in higher organisms, specifically the Nav 1.4 and Nav 1.5 channel subtypes. However, the native physiological function that the brevetoxins perform for K. brevis is unknown. By using fluorescent and photoactivatable derivatives, brevetoxin was shown to localize to the chloroplast of K. brevis where it binds to the light-harvesting complex II (LHCII) and thioredoxin. The LHCII is essential to non-photochemical quenching (NPQ), whereas thioredoxins are critical to the maintenance of redox homeostasis within the chloroplast and contribute to the scavenging of reactive oxygen. A culture of K. brevis producing low levels of toxin was shown to be deficient in NPQ and produced reactive oxygen species at twice the rate of the toxic culture, implicating a role in NPQ for the brevetoxins.

Synthesis, receptor binding and activity of iso and azakainoids.

Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction of the 1-pyrazoline NN bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting 2-pyrazoline obtained after desilylation may be reduced with NaCNBH3 to provide the trans azakainate analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described. Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid demonstrates that it is an ionotropic glutamate receptor agonist.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.

Biosynthesis of Athmu, a α,γ-hydroxy-ß-amino acid of pahayokolides A-B.

Pahayokolides A-B are cyanobacteria derived non-ribosomal peptides which exhibit cytotoxicity against a number of cancer cell lines. The biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu) moiety has been investigated using stable isotope incorporation experiments. While α-ketoisocaproic acid (α-KIC), α-hydroxyisocaproic acid (α-HIC) and leucine all serve as precursors to Athmu, the feeding of [1-(13)C] α-KIC results in more than threefold greater (13)C enrichment than the other precursors. This result suggests that α-KIC is the immediate precursor which is selected and activated by the adenylation domain of the loading NRPS module and subsequently reduced in a fashion similar to that of the recently identified pathways for cryptophycins A-B, cereulide and valinomycin.

GENE EXPRESSION PROFILING OF HUMAN LIVER CARCINOMA (HepG2) CELLS EXPOSED TO THE MARINE TOXIN OKADAIC ACID.

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.

The structures of three metabolites of the algal hepatotoxin okadaic acid produced by oxidation with human cytochrome P450.

Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 µg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,ß unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A.

Biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid moiety and absolute configuration of pahayokolides A and B.

Pahayokolides A and B are cyclic undecapeptides that were isolated from the cyanobacterium Lyngbya sp. They contain the unusual α-hydroxy-ß-amino acid 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configurations of the amino acids of the pahayokolides, except for the four oxygen-bearing stereocenters of Athmu, have been determined by Marphy's method. Incorporation of labeled leucine and acetate precursors into the pahayokolides has established that Athmu is derived from a leucine or α-keto isocaproic acid starter unit, which is further extended with three acetate units.

Biodegradation of polyether algal toxins--isolation of potential marine bacteria.

Marine algal toxins such as brevetoxins, okadaic acid, yessotoxin, and ciguatoxin are polyether compounds. The fate of polyether toxins in the aqueous phase, particularly bacterial biotransformation of the toxins, is poorly understood. An inexpensive and easily available polyether structural analog salinomycin was used for enrichment and isolation of potential polyether toxin degrading aquatic marine bacteria from Florida bay area, and from red tide endemic sites in the South Florida Gulf coast. Bacterial growth on salinomycin was observed in most of the enrichment cultures from both regions with colony forming units ranging from 0 to 6×10(7) per mL. The salinomycin biodegradation efficiency of bacterial isolates determined using LC-MS ranged from 22% to 94%. Selected bacterial isolates were grown in media with brevetoxin as the sole carbon source to screen for brevetoxin biodegradation capability using ELISA. Out of the two efficient salinomycin biodegrading isolates MB-2 and MB-4, maximum brevetoxin biodegradation efficiency of 45% was observed with MB-4, while MB-2 was unable to biodegrade brevetoxin. Based on 16S rRNA sequence similarity MB-4 was found have a match with Chromohalobacter sp.

Human metabolites of brevetoxin PbTx-2: Identification and confirmation of structure.

Four metabolites were identified upon incubation of brevetoxin (PbTx-2) with human liver microsomes. Chemical transformation of PbTx-2 confirmed the structures of three known metabolites BTX-B5, PbTx-9 and 41, 43-dihydro-BTX-B5 and a previously unknown metabolite, 41, 43-dihydro-PbTx-2. These metabolites were also observed upon incubation of PbTx-2 with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Cytochrome P450 3A4 produced oxidized metabolites while other CYPs generated the reduced products.

New peptides isolated from Lyngbya species: a review.

Cyanobacteria of the genus Lyngbya have proven to be prodigious producers of secondary metabolites. Many of these compounds are bioactive and show potential for therapeutic use. This review covers peptides and hybrid polyketide-non-ribosomal peptides isolated from Lyngbya species. The structures and bioactivities of 50 Lyngbya peptides which were reported since 2007 are presented.

The algal hepatoxoxin okadaic acid is a substrate for human cytochromes CYP3A4 and CYP3A5.

The hepatotoxin okadaic acid (OA) was incubated with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Both CYP3A4 and CYP3A5 converted OA to a mixture of the same four metabolites, but incubation with CYP3A4 resulted in higher levels of conversion. Michaelis-Menten parameters, K(m) (73.4 microM) and V(max) (7.23 nmol of metabolitesnmol(-1)min(-1)) for CYP3A4 were calculated by analyzing double-reciprocal plots. LC-MS(n) analysis and chemical interconversion indicate that metabolites 2 and 3 are the 11S-hydroxy and 11R-hydroxy okadaic acid respectively, while metabolite 4 is 11-oxo okadaic acid. LC-MS(n) analysis of metabolite 1 shows a molecular ion which corresponds to an addition of 16 amu to OA, also suggesting hydroxylation, but the specific site has not been identified. The same four metabolites were produced upon incubation of okadaic acid with pooled human liver microsomes. This transformation could be completely inhibited with ketokonazole, and inhibitor of the CYP3A family of enzymes. The metabolites were determined to be only slightly less potent inhibitors of serine threonine protein phosphatase 2A (PP2A) when compared to OA. As PP2A is the principle molecular target for OA, these oxidative transformations may not effectively detoxify OA.