Stephan Gromer

Human selenoproteins at a glance

Abstract.The public perception of selenium has changed significantly over the last decades. Originally mainly known for its high toxicity, it was later recognized as an essential trace element and is now (despite its narrow therapeutic window) almost being marketed as a lifestyle drug. Indeed, some clinical and preclinical studies suggest that selenium supplementation may be beneficial in a large number of clinical conditions. However, its mode of action is unresolved in most of these cases. Selenocysteine – identified as the 21st amino acid used in ribosome-mediated protein synthesis – is incorporated in at least 25 specific, genetically determined human selenoproteins, many of which have only recently been discovered. Restoration of normal selenoprotein levels may be – apart from direct supranutritional effects – one possible explanation for the effects of selenium supplements. In this review we provide a brief but up-to-date overview of what is currently known about these 25 acknowledged human selenoproteins and their synthesis.

Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum.

ABSTRACT Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M−1 s−1), thioredoxins (k = 8.5 to 26 M−1 s−1), dihydrolipoamide (k = 53 M−1 s−1), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the kcat values ranging from 0.03 s−1 to 10 s−1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.

Antioxidant Status and Nitric Oxide in the Malnutrition Syndrome Kwashiorkor

The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still poorly understood. The syndrome is, however, known to be associated with alterations in redox metabolism. To further elucidate the role of oxidative stress in kwashiorkor, we carried out a longitudinal study on the major blood antioxidants at the St. Josephs Hospital, Jirapa, Ghana. All kwashiorkor patients (K) were followed up for 20 d. In comparison with local healthy controls (C), the plasma total antioxidant status was reduced to less than 50% in the patients (C, 0.87 ± 0.21 mM; K, 0.40 ± 0.20 mM;p < 0.001). Similarly, the major plasma antioxidant albumin (C, 40.9 ± 2.5 g/L; K, 19.1 ± 7.4 g/L;p < 0.001) and erythrocyte glutathione (C, 2.39 ± 0.28 mM; K, 1.01 ± 0.33;p < 0.001) were decreased, whereas the levels of bilirubin and uric acid were not significantly altered. Nitrite and nitrate were found to be increased by a factor of 2 in kwashiorkor (C, 120 ± 46 μM; K, 235 ± 107 μM;p < 0.001). Over the observation period, the trends of albumin and glutathione levels were related to clinical outcome. These concentrations rose in patients who recovered and fell in patients who did not. Our study strongly supports the hypothesis that oxidative and nitrosative stress play a role in the pathophysiology of edematous malnutrition. Prophylactic and therapeutic strategies should aim at the careful correction of the reduced antioxidant status of the patients.

Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein

Proteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with 75Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins.

The 58 kDa mouse selenoprotein is a BCNU-sensitive thioredoxin reductase

The flavoprotein thioredoxin reductase [EC 1.6.4.5] (NADPH+H++thioredoxin‐S2→NADP++thioredoxin‐(SH)2) was isolated from mouse Ehrlich ascites tumour (EAT) cells. Like the counterpart from human placenta but unlike the known thioredoxin reductases from non‐vertebrate organisms, the mouse enzyme was found to contain 1 equivalent of selenium per subunit of 58 kDa. The K M values were 4.5 μM for NADPH, 480 μM for DTNB and 36 μM for Escherichia coli thioredoxin, the turnover number with DTNB being ≈40 s−1. As mouse is a standard animal model in cancer and malaria research, thioredoxin reductase and glutathione reductase [EC 1.6.4.2] from EAT cells were compared with each other. While both enzymes in their 2‐electron reduced form are targets of the cytostatic drug carmustine (BCNU), no immunologic cross‐reactivity between the two mouse disulfide reductases was observed.

Mutational Studies Confirm the Catalytic Triad in the Human Selenoenzyme Thioredoxin Reductase Predicted by Molecular Modeling

The glutathione and the thioACHTUNGTRENNUNGredoxin systems are key players in the cellular redox milieu in almost all organisms, and are, therefore, well established drug targets for a considerable number of clinical conditions. In both systems, NADPH-dependent flavoenzymes—namely glutathione reductase (GR; EC 1.8.1.7) and thioredoxin reductase (TrxR; EC 1.8.1.9)—are involved. Both enzymes belong to a family of homodimeric pyridine nucleotide-disulfide oxidoACHTUNGTRENNUNGreductases, which includes enzymes like lipoamide dehydrogenase, trypanothione reductase, and mercuric ion reductase. Two very distinct classes of thioredoxin reductases exist : the so called small (subunit Mr~ 35 kDa) and large (subunit Mr~ 55 kDa) thioredoxin reductases. Structure and catalytic mechanism of small TrxRs—present primarily in bacteria, fungi, and plants—are known in atomic detail, whereas many aspects regarding their larger counterparts are still unresolved. Mammalian thioredoxin reductases are large TrxRs and have gathered significant scientific attention in recent years as they are members of the small and elite selenoenzyme family. 4] Thus, they are potential mediators for the clinically observed beneficial effects of selenium supplements and also interesting drug targets. 5, 6] Selenocysteine (Sec) is the penultimate amino acid in each subunit’s polypeptide chain and is located on a flexible C-terminal tail. 8] The currently accepted model for the catalytic mechanism of these enzymes involves the transfer of electrons from NADPH’s reduced nicotinamide ring via flavin (FAD) to the N-terminal cysteines (Cys59 and Cys64). Here the electrons are thought to be picked up by the second subunit’s C-terminal redox active site, which is formed by the adjacent Cys497– Sec498 pair, and subsequently transferred to the final substrate, for example, thioredoxin (Scheme 1 A). Both, dithiol–disulfide and thiol/selenol–selenenylsulfide exchange reactions typically require the formation of a thiolate

The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue

Abstract The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.

Cytotoxic interactions of methylene blue with trypanosomatid-specific disulfide reductases and their dithiol products.

Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.

Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum

ABSTRACT 4′,5′-Dibromo-2′,7′-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC50) of 180 μM. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC50 of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.

The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function

The catalytic activity of selenocysteine‐containing thioredoxin reductases can be mimicked by cysteine‐variants if the local environment at the C‐terminal redox center supports thiol activation. This concept of a linear catalytic site was challenged by structural data suggesting that the invariant residue His106 functions as a base catalyst for the dithiol‐disulphide exchange reaction between enzyme and substrate. As reported here, we changed His106 to asparagine, glutamine, and phenylalanine in various C‐terminal mutants of Drosophila melanogaster thioredoxin reductase. The catalytic activity dropped considerably, yet pH‐profiles did not reveal differences, rendering a function for His106 as a base catalyst unlikely. Interestingly, the phenylalanine‐mutants, designed as negative controls were the most active mutants which suggests rather a structural role of His106.