Peter Steinert

GLUTATHIONE AND TRYPANOTHIONE IN PARASITIC HYDROPEROXIDE METABOLISM

Thiol-dependent hydroperoxide metabolism in parasites is reviewed in respect to potential therapeutic strategies. The hydroperoxide metabolism of Crithidia fasciculata has been characterized to comprise a cascade of three enzymes, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase, plus two supportive enzymes to synthesize the redox mediator trypanothione from glutathione and spermidine. The essentiality of the system in respect to parasite vitality and virulence has been verified by genetic approaches. The system appears to be common to all genera of the Kinetoplastida. The terminal peroxidase of the system belongs to the protein family of peroxiredoxins which is also represented in Entamoeba and a variety of metazoan parasites. Plasmodial hydroperoxide metabolism displays similarities to the mammalian system in comprising glutathione biosynthesis, glutathione reductase, and at least one glutathione peroxidase homolog having the active site selenocysteine replaced by cysteine. Nothing precise is known about the antioxidant defence systems of Giardia, Toxoplasma, and Trichomonas species. Also, the role of ovothiols and mycothiols reportedly present in several parasites remains to be established. Scrutinizing known enzymes of parasitic antioxidant defence for suitability as drug targets leaves only those of the trypanosomatid system as directly or indirectly validated. By generally accepted criteria of target selection and feasibility considerations tryparedoxin and tryparedoxin peroxidase can at present be rated as the most appealing target structures for the development of antiparasitic drugs.

Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation

Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142–6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin‐dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage‐specific expression in late spermatids, which are under the control of Leydig cell‐derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.—Maiorino, M., Wissing, J. B., Brigelius‐ Flohe´, R., Calabrese, F., Roveri, A., Steinert, P., Ursini, F., Flohe´, L. Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation. FASEB J. 12, 1359–1370 (1998)

Bmp-2 downstream targets in mesenchymal development identified by subtractive cloning from recombinant mesenchymal progenitors (C3H10T½)†

A Bmp‐dependent in vitro model was used to identify cDNAs during the manifestation of mesenchymal lineages. This model involves the recombinant expression of Bmps (Bmp‐2, Bmp‐4–7) in murine mesenchymal C3H10T½ progenitors, which leads to the differentiation into three lineages: the osteogenic, the chondrogenic and the adipogenic lineage, albeit in varying efficiencies. By subtractive cloning, 21 Bmp‐2–regulated cDNAs from C3H10T½ mesenchymal progenitors were identified; 20 were related to known sequences and 1 was not. During mouse embryonic development, many of these cDNAs are expressed in chondrogenic, osteogenic, and in adipogenic tissues. Novel findings include a G0/G1 switch gene (G0S2), which was demonstrated to be predominantly expressed in adipose tissue during late murine embryonic development. Furthermore, the membrane‐standing glycoprotein autotaxin (ATX) is expressed, at precartilage condensations, joint regions, and during tooth development. An as yet undescribed cDNA, 29A, which encodes a putative secreted factor, is expressed in developing osteo‐/chondrogenic tissues of vertebrae, ribs, tooth, and the limb bud. C3H10T½‐progenitors, therefore, may serve as a legitimate model for the investigation of the Bmp‐mediated events during mesenchymal differentiation. Dev. Dyn. 1998;213:398–411.

Structures of tryparedoxins revealing interaction with trypanothione.

Abstract Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin type peroxidases by the bisglutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3Dstructures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the histagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solventexposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the [1]Nglutathionyl residue of trypanothione, as evidenced by the structure of [1]Nglutathionylspermidinederivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cisPro111 (110)bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXNsubstrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the [1]Nglutathionylspermidine binding in the CfTXN2H6-Cys44Ser structure.

Analysis of the mouse selenoprotein P gene.

In vertebrates several proteins containing a covalently bound selenocysteine residue have been identified. Among these, selenoprotein P is the most unusual one: depending on the species, 8-12 selenocysteine residues are cotranslationally integrated into the polypeptide chain. The protein was traced in rat plasma, but its role has not been worked out so far. In order to improve our understanding on selenoprotein P we investigated its tissue-specific expression and its genomic DNA. RNA in situ hybridization analyses confirmed the liver-specific expression in mice. Selenoprotein P was also found to be expressed in testis, brain, gut, and hematopoietic cells. The murine selp gene contains five exons within 10.3 kb with a coding sequence restricted to exons 2 to 5. The complete gene including the selp promoter was sequenced. One TATA motif 38 bp upstream to exon 1 suggests transcription of selp by RNA polymerase II. Within the 1116 bp upstream of exon 1 four hepatic nuclear factor 3beta (HNF3beta) binding motifs were found, which is in line with liver-specific expression of selenoprotein P. The expression in hematopoietic cells might be due to multiple GATA-1 motifs. Two BRN-2 motifs suitable for the binding of brain-specific regulatory factors correlated to the selenoprotein P expression in the cerebellum. Selenoprotein P was also expressed in Leydig cells which could be regulated by binding proteins docking to the SRY motifs present in the promoter region.

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening.

Abstract Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A600 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a kcat of 1.7 s−1 and limiting Km values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively.

SEQUENCE, HETEROLOGOUS EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF A NOVEL TRYPAREDOXIN FROM CRITHIDIA FASCICULATA

Tryparedoxin has recently been discovered as a constituent of the trypanosomal peroxidase system catalysing the reduction of a peroxiredoxin-type peroxidase by trypanothione [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] and has attracted interest as a potential molecular target for the development of trypanocidal agents. Here we describe the first isolation of a novel gene from Crithidia fasciculata encoding a different tryparedoxin designated tryparedoxin II. The deduced amino acid sequence of tryparedoxin II (accession number AF055986) differs substantially from the partial sequence reported for the tryparedoxin described previously and now renamed tryparedoxin I. It shares the sequence motif Vx3FSAxWCPPCR shown to represent the catalytic site in tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918] with mouse nucleoredoxin (accession number X92750), and a thioredoxin-like gene product of Caenorhabditis elegans (accession number U23511). Depending on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger than the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagged tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (KM peroxiredoxin = 4.2 microM, KM trypanothione = 33 microM, Vmax/[E] = 952 min(-1)) similar to those reported for tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918]. The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.

Cytoplasmic localization of the trypanothione peroxidase system in Crithidia fasciculata.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.

Permutation of the active site motif of tryparedoxin 2.

Abstract Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.

Manganese-stimulated phosphatidylinositol 4-kinase from Dunaliella parva: purification and characterization☆

Abstract A soluble phosphatidylinositol (PI) kinase (ATP:phosphatidylinositol phosphotransferase, EC 2.7.1.67) was partially purified from Dunaliella parva. Analyses of the enzyme product and its water-soluble moiety, obtained by deacylation and removal of the glycerol, revealed that the enzyme is a PI 4-kinase. An apparent molecular weight of about 500 000 was determined by size exclusion chromatography and glycerol density gradient centrifugation. Chromatofocusing and isoelectric focusing displayed pI values of 5.5 and 6.6, respectively. The kinase required divalent cations such as Mg2+ (optimum at 30 mM) and Ca2+ (optimum at 10 mM); however, in the presence of Mn2+ ions (optimum at 10 mM) the activity was about 2.5-fold higher. Furthermore, Mn2+ and Mg2+ (or Ca2+) ions activated synergistically; at a constant Mg2+ concentration (2 mM), Mn2+ ions stimulated the activity up to about 20-fold, whereas at a constant Mn2+ concentration (10 mM) Mg2+, ions stimulated up to about 4-fold. The temperature optimum of the enzyme activity increased from 28°C at 30 mM MgCl2 to 42°C at 2 mM MgCl2 and 10 mM MnCl2, whereas the pH optimum of 7.8 did not change. The apparent Km value for ATP depended on the divalent cation; at 2 mM MgCl2 and 10 mM MnCl2, a Km value of 390 μM was determined, whereas at 30 mM MgCl2 the value was 1.7 mM. The activity of the lipid kinase depended on the presence of a surfactant; optimum concentration of Triton X-100 was 3.7 mM (235 μM PI, 2 mM MgCl2 and 10 mM MnCl2). The apparent Km value for PI was determined as 55 and 27 μM at 1.5 and 0.75 mM Triton X-100, respectively, which corresponds to a surface concentration of PI in the mixed micelles of about 3.6 mol%. Several nucleoside triphosphates as well as ADP, AMP, adenosine and phosphatidylethanolamine were shown to inhibit the enzyme competitively.