14 research outputs found
The 58 kDa mouse selenoprotein is a BCNU-sensitive thioredoxin reductase
No full textAbstractThe 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 KM 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
The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function
No full textAbstractThe 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
Mutational Studies Confirm the Catalytic Triad in the Human Selenoenzyme Thioredoxin Reductase Predicted by Molecular Modeling
No full textSelenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein
No full textProteins 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
Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium falciparum
No full textMethylene 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 M1 s1), thioredoxins (k 8.5 to 26 M1 s1), dihydrolipoamide (k 53 M1 s1), 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 s1 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. Thi
Synthesis of 5-Nitro-2-furancarbohydrazides and Their <i>cis</i>-Diamminedichloroplatinum Complexes as Bitopic and Irreversible Human Thioredoxin Reductase Inhibitors
No full textThe human selenoprotein thioredoxin reductase is involved in antioxidant defense and DNA
synthesis. As increased thioredoxin reductase levels are associated with drug sensitivity to
cisplatin and drug resistance in tumor cells, this enzyme represents a promising target for the
development of cytostatic agents. To optimize the potential of the widely used cisplatin to inhibit
the human thioredoxin reductase and therefore to overcome cisplatin resistance, we developed
and synthesized four cis-diamminedichloroplatinum complexes of the lead 5-nitro-2-furancarbohydrazide 8 selected from high-throughput screening. Detailed kinetics revealed that the
isolated fragments, 5-nitro-2-furancarbohydrazide and cisplatin itself, bind with micromolar
affinities at two different subsites of the human enzyme. By tethering both fragments four
nitrofuran-based cis-diamminedichloroplatinum complexes 13a−c and 20 were synthesized
and identified as biligand irreversible inhibitors of the human enzyme with nanomolar affinities.
Studies with mutant enzymes clearly demonstrate the penultimate selenocysteine residue as
the prime target of the synthesized cis-diamminedichloroplatinum complexes
The \u3ci\u3ePlasmodium\u3c/i\u3e selenoproteome
No full textThe use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial parasites on selenium suggests possible strategies for antimalarial drug development
