DOCSLIB.ORG

Glutathione and the Redox Control System Trypanothione/Trypanothione Reductase Are Involved in the Protection of Leishmania Spp

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Glutathione and the Redox Control System Trypanothione/Trypanothione Reductase Are Involved in the Protection of Leishmania Spp Brazilian Journal of Medical and Biological Research (2006) 39: 355-363 Protection of Leishmania by glutathione/trypanothione systems 355 ISSN 0100-879X Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity P.R.T. Romão1, 1Laboratório de Imunoparasitologia, Curso de Medicina, J. Tovar2, S.G. Fonseca3, Universidade do Sul de Santa Catarina (UNISUL), Tubarão, SC, Brasil R.H. Moraes4, 2School of Biological Sciences, Royal Holloway University of London, Egham, UK A.K. Cruz5, J.S. Hothersall6, 3Laboratório de Imunologia, Instituto do Coração, Faculdade de Medicina, A.A. Noronha-Dutra6, Universidade de São Paulo, São Paulo, SP, Brasil S.H. Ferreira4 4Departamento de Farmacologia, 5Departamento de Biologia Celular e Molecular e and F.Q. Cunha4 Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, de São Paulo, Ribeirão Preto, SP, Brasil 6The Institute of Urology and Nephrology, University College of London, London, UK Abstract Correspondence Glutathione is the major intracellular antioxidant thiol protecting Key words P.R.T. Romão mammalian cells against oxidative stress induced by oxygen- and • Leishmania Laboratório de Imunoparasitologia nitrogen-derived reactive species. In trypanosomes and leishmanias, • Glutathione Curso de Medicina, UNISUL trypanothione plays a central role in parasite protection against mam- • Trypanothione Rua José Acácio Moreira, 787, malian host defence systems by recycling trypanothione disulphide by • Trypanothione reductase DEHON • the enzyme trypanothione reductase. Although Kinetoplastida para- Nitric oxide 88704-900 Tubarão, SC • Free radicals Brasil sites lack glutathione reductase, they maintain significant levels of Fax: +55-48-621-3067 glutathione. The aim of this study was to use Leishmania donovani E-mail: [email protected] trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the Research supported by FAPESP protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (No. 97/01938-8). P.R.T. Romão and (SNAP), a nitrogen-derived reactive species donor. We found that the S.G. Fonseca were recipients of FAPESP fellowships. F.Q. Cunha, resistance to SNAP of different species of Leishmania was inversely S.H. Ferreira, and R.H. Moraes were correlated with their glutathione concentration but not with their total 7 recipients of CNPq fellowships. low-molecular weight thiol content (about 0.18 nmol/10 parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/107 parasites, respectively. L. amazonensis, Received January 21, 2005 that have a higher level of glutathione, were less susceptible to SNAP Accepted November 11, 2005 (30 and 100 µM). The IC50 values of SNAP determined to L. amazo- nensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a de- creased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, par- ticipate in protection of Leishmania against the toxic effect of nitro- gen-derived reactive species. Braz J Med Biol Res 39(3) 2006 356 P.R.T. Romão et al. Introduction plays a crucial role in the killing of parasites both in vitro (7) and in vivo (8,9). In vitro The trypanosomatids, members of the or- macrophage microbicidal activity correlates der Kinetoplastida, include parasitic protozoa with NO production, and both in vivo and in of importance to public health such as Leish- vitro microbicidal activities are completely mania spp. Leishmania cause a spectrum of inhibited by the NO synthase inhibitor L- diseases ranging from self-healing ulcers to arginine analogue NG-monomethyl-L-argi- disseminated and often fatal infections, de- nine (L-NMMA) but not by its enantiomer pending on the species involved and the host’s D-NMMA (8). In addition, the NO donors, immune response. Adequate vaccines against S-nitroso-N-acetyl-D,L-penicillamine (SNAP) trypanosomatid infections have yet to be de- and 3-morpholino-sydnonimine hydrochlo- veloped, and drugs currently available for che- ride are able to kill Leishmania parasites in a motherapeutic intervention are mostly unsat- cell-free model system (10). isfactory mainly because of their lack of speci- We have reported that glutathione is in- ficity, toxicity to humans, and, in many cases, volved in the protection of mammalian mac- developed parasite resistance (1). Thus, one of rophages against the cytotoxic effects of the priorities in tropical medicine research has NO. Furthermore, despite evidence that glu- been the identification and characterisation of tathione in Leishmania appears not to be the parasite-specific biomolecules, which play major antioxidant, we have demonstrated relevant physiological roles and thus might be that it protects L. major from the toxic ef- exploited as selective targets. fects of NO (11). Among many other metabolic distinctions, In the present study, we have extended trypanosomatids maintain their intracellular these observations by comparing the glutathi- redox balance by a mechanism that is different one levels and SNAP sensitivity of different from that of their insect vectors and mamma- Leishmania species: L. amazonensis, L. brazi- lian hosts. They lack glutathione reductase, liensis, L. donovani, and L. major. Our results which in nearly all other organisms is respon- demonstrate that the sensitivity of distinct spe- sible for the maintenance of an intracellular cies of Leishmania to SNAP is inversely cor- thiol-reducing environment, and thus for the related with their glutathione concentration. reduction of disulphides, detoxification of per- When we extended our investigation to the oxides and synthesis of DNA precursors (2). role of TryR in the protection against SNAP Instead, they possess a unique system using using mutants of L. donovani for the TryR trypanothione [T(SH)2] that is the major re- gene (tryR, formerly tryA) (12), we found duced thiol of Kinetoplastida parasites (3) and that, compared to control parasites (tryR geno- comprises a spermidine moiety linked to two type+/+/+), a double mutant clone (tryR geno- glutathione molecules (2). Together with three type-/-/+) was significantly more sensitive to thiol-redox proteins, trypanothione reductase the cytotoxic effect of SNAP. Overall, these (TryR), tryparedoxin and tryparedoxin per- results demonstrate that glutathione as well as oxidase (4,5), T(SH)2 is thought to provide the T(SH)2/TryR redox system are essential defence against oxidants, certain heavy metals protective components against NO cytotoxic- (6) and xenobiotics (3). Thus, TryR has a vital ity in Leishmania. physiological role in maintaining T(SH)2 re- dox, particularly within the highly oxidative Material and Methods intracellular environment of the host cells which is generated during the antimicrobial Leishmania strains and culture conditions defence response. In murine leishmaniasis, nitric oxide (NO) The Leishmania species used in this study Braz J Med Biol Res 39(3) 2006 Protection of Leishmania by glutathione/trypanothione systems 357 were L. braziliensis (MHOM/BR/75/M2904), ols and glutathione (reduced plus disulphide L. amazonensis (MPRO/BR/72/M1841-LV- forms) were measured in lysates of promas- 79), L. major (LV-39, clone 5-Rho-SU/59/P), tigote forms (stationary phase) of different and L. donovani (clone LV9-3 from MHOM/ Leishmania species (L. amazonensis, L. bra- ET/67/HU3). The L. donovani clones used ziliensis, L. donovani, and L. major) includ- were: wild-type LV9-3, which possesses ing the different clones of L. donovani. Low- three copies of the tryR (formerly tryA) gene molecular weight thiols were measured us- (tryR+/+/+) and the mutants of TryR: clones ing Ellman’s reagent (15). To measure H2-tryR-/+/+ (LV9-3 submitted to single re- soluble thiols the samples were deproteinised placement), clone HB3-tryR-/-/+ (LV9-3 sub- with 1% sulfosalicylic acid in the presence mitted to double replacement (12) and clone of 5 mM EDTA. The concentration of SH HB3-pTTcTR (Tovar J and Fairlamb AH, groups was calculated from a standard curve unpublished results) that is identical to clone of 0.01 to 2 nmol cysteine. Glutathione lev- HB3 (tryR-/-/+) but harbours plasmid pTTcTR els were measured by the glutathione reduc- (13). Promastigote forms of all Leishmania tase enzyme recycling method (16). These species were grown in M199 medium supple- assays were adapted for use in a microtitre mented with 40 mM HEPES, pH 7.4, 0.1 mM plate using a microplate spectrophotometer adenine, 7.7 mM hemin, 10% (v/v) heat-inac- system spectra MAX 250 (Molecular De- tivated foetal calf serum, 50 U/mL penicillin, vices, Union City, CA, USA). Cells were and 50 µg/mL streptomycin. Cultures were lysed by the addition of 100 µL 1 mM EDTA incubated at 26ºC, and cells were kept at to each well and freezing immediately. Fol- densities ranging between 5 x 105 and 3 x 107 lowing thawing, plates were shaken for 30 s parasites/mL. Transfectants were cultured in and then sonicated for 60 s. Assays were the presence of selective drugs (12). The mu- carried out immediately (17). Although this tant H2-tryR-/+/+ was cultured in the presence assay provides a measure of both oxidised of 16 µg/mL hygromycin B, the clone HB3- and reduced glutathione, in non-oxidative tryR-/-/+ in the presence of 16 µg/mL stress equilibrium, the cellular condition hygromycin B plus 2.5 µg /mL phleomycin, under which we have measured glutathione, and the HB3-tryR-/-/+ clone plus episomal the thiol content is 95-99% reduced glutathi- pTTcTR in the presence of 16 µg/mL one. hygromycin B, 25 µg/mL G418 and 2.5 µg/ mL phleomycin. Cytotoxic effect of S-nitroso-N-acetyl-D,L- penicillamine on Leishmania viability Growth curves The direct cytotoxic effect of the nitroso- Promastigotes of wild-type or L.
Load more

Recommended publications
  • Diverse Biosynthetic Pathways and Protective Functions Against Environmental Stress of Antioxidants in Microalgae
    plants Review Diverse Biosynthetic Pathways and Protective Functions against Environmental Stress of Antioxidants in Microalgae Shun Tamaki 1,* , Keiichi Mochida 1,2,3,4 and Kengo Suzuki 1,5 1 Microalgae Production Control Technology Laboratory, RIKEN Baton Zone Program, Yokohama 230-0045, Japan; [email protected] (K.M.); [email protected] (K.S.) 2 RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan 3 Kihara Institute for Biological Research, Yokohama City University, Yokohama 230-0045, Japan 4 School of Information and Data Sciences, Nagasaki University, Nagasaki 852-8521, Japan 5 euglena Co., Ltd., Tokyo 108-0014, Japan * Correspondence: [email protected]; Tel.: +81-45-503-9576 Abstract: Eukaryotic microalgae have been classified into several biological divisions and have evo- lutionarily acquired diverse morphologies, metabolisms, and life cycles. They are naturally exposed to environmental stresses that cause oxidative damage due to reactive oxygen species accumulation. To cope with environmental stresses, microalgae contain various antioxidants, including carotenoids, ascorbate (AsA), and glutathione (GSH). Carotenoids are hydrophobic pigments required for light harvesting, photoprotection, and phototaxis. AsA constitutes the AsA-GSH cycle together with GSH and is responsible for photooxidative stress defense. GSH contributes not only to ROS scavenging, but also to heavy metal detoxification and thiol-based redox regulation. The evolutionary diversity of microalgae influences the composition and biosynthetic pathways of these antioxidants. For example, α-carotene and its derivatives are specific to Chlorophyta, whereas diadinoxanthin and fucoxanthin are found in Heterokontophyta, Haptophyta, and Dinophyta. It has been suggested that Citation: Tamaki, S.; Mochida, K.; Suzuki, K. Diverse Biosynthetic AsA is biosynthesized via the plant pathway in Chlorophyta and Rhodophyta and via the Euglena Pathways and Protective Functions pathway in Euglenophyta, Heterokontophyta, and Haptophyta.
    [Show full text]
  • Development of Drug Resistance in Trypanosoma Brucei Rhodesiense and Trypanosoma Brucei Gambiense
    411-419 13/9/08 11:56 Page 411 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 22: 411-419, 2008 411 Development of drug resistance in Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Treatment of human African trypanosomiasis with natural products (Review) STEFANIE GEHRIG1 and THOMAS EFFERTH2 1Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 2German Cancer Research Center, Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received April 10, 2008; Accepted June 12, 2008 DOI: 10.3892/ijmm_00000037 Abstract. Human African trypanosomiasis is an infectious Contents disease which has resulted in the deaths of thousands of people in Sub-Saharan Africa. Two subspecies of the 1. Introduction protozoan parasite Trypanosoma brucei are the causative 2. Disease and clinical manifestation agents of the infection, whereby T. b. gambiense leads to 3. Standard chemotherapy chronic development of the disease and T. b. rhodesiense 4. Development of resistance establishes an acute form, which is fatal within months or 5. Screening of natural products even weeks. Current chemotherapy treatment is complex, 6. Conclusion since special drugs have to be used for the different development stages of the disease, as well as for the parasite concerned. Melarsoprol is the only approved drug for 1. Introduction effectively treating both subspecies of human African trypanosomiasis in its advanced stage, however, the drug's Human African typanosomiasis is a vector-born parasitic potency is constrained due to an unacceptable side effect: disease demonstrating a major public health problem for encephalopathy, which develops in one out of every 20 people in the Sub-Saharan region.
    [Show full text]
  • A Tryparedoxin-Coupled Biosensor Reveals a Mitochondrial Trypanothione Metabolism in Trypanosomes
    1 A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione 2 metabolism in trypanosomes 3 Samantha Ebersoll1, Marta Bogacz1, Lina M. Günter1, Tobias P. Dick2, R. Luise 4 Krauth-Siegel1* 5 1 6 Biochemie-Zentrum der Universität Heidelberg, Heidelberg, Germany; 2Division of Redox 7 Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, 8 Germany 9 10 11 *For correspondence: [email protected] 12 13 14 15 Competing interests: 16 The authors declare that no competing interests exist. 1 17 Abstract Trypanosomes have a trypanothione redox metabolism that provides the reducing 18 equivalents for numerous essential processes, most being mediated by tryparedoxin (Tpx). 19 While the biosynthesis and reduction of trypanothione are cytosolic, the molecular basis of 20 the thiol redox homeostasis in the single mitochondrion of these parasites has remained 21 largely unknown. Here we expressed Tpx-roGFP2, roGFP2-hGrx1 or roGFP2 in either the 22 cytosol or mitochondrion of Trypanosoma brucei. We show that the novel Tpx-roGFP2 is a 23 superior probe for the trypanothione redox couple and that the mitochondrial matrix harbors a 24 trypanothione system. Inhibition of trypanothione biosynthesis by the anti-trypanosomal drug 25 Eflornithine impairs the ability of the cytosol and mitochondrion to cope with exogenous 26 oxidative stresses, indicating a direct link between both thiol systems. Tpx depletion abolishes 27 the cytosolic, but only partially affects the mitochondrial sensor response to H2O2. This 28 strongly suggests that the mitochondrion harbors some Tpx and, another, as yet unidentified, 29 oxidoreductase. 30 31 Introduction 32 Trypanosomatids are the causative agents of African sleeping sickness (Trypanosoma brucei 33 gambiense and T.
    [Show full text]
  • Trypanosomatid Hydrogen Peroxidase Metabolism
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 221, number 2, 427-431 FEB 05098 September 1987 Trypanosomatid hydrogen peroxidase metabolism P.G. Penketh, W.P.K. Kennedy*, C.L. Patton and A.C. Sartorelli MacArthur Center for Parasitology and Tropical Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA and * Wolfson Molecular Biology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WCLE 7HT, England Received 7 July 1987 The rate of whole cell HZ02 metabolism in several salivarian and stercorarian trypanosomes and Leishrnania species was measured. These cells metabolized Hz02 at rates between 2.3 and 48.2 nmol/lO* cells per min depending upon the species employed. Hz02 metabolism was largely insensitive to NaN3, implying that typi- cal catalase and peroxidase haemoproteins are not important in Hz02 metabolism. The metabolism of H202, however, was almost completely inhibited by N-ethylmaleimide. In representative species, Hz02 metabolism was shown to occur through a trypanothione-dependent mechanism. Hydrogen peroxide; Trypanothione; Macrophage; (Trypanosoma, Leishmania) 1. INTRODUCTION been reported that L. donovani possesses a surface-membrane acid phosphatase which reduces Many trypanosomatids have been reported to the magnitude of the oxidative burst of lack or to be extremely deficient in enzyme systems neutrophils, inferring a pathophysiological role for necessary for the removal of Hz02 (i.e. catalase, this enzyme [5]. However, defense mechanisms glutathione peroxidase) [l-5]. Defense against endogenously generated Hz02 and the mechanisms against H202, however, appear to be possibly diminished quantity of Hz02 produced a ubiquitous requirement of most aerobic cells [6].
    [Show full text]
  • Epigallocathechin-O-3-Gallate Inhibits Trypanothione Reductase of Leishmania Infantum, Causing Alterations in Redox Balance And
    ORIGINAL RESEARCH published: 25 March 2021 doi: 10.3389/fcimb.2021.640561 Epigallocathechin-O-3-Gallate Inhibits Trypanothione Reductase of Leishmania infantum, Causing Alterations in Redox Balance and Edited by: Leading to Parasite Death Brice Rotureau, Institut Pasteur, France Job D. F. Inacio 1, Myslene S. Fonseca 1, Gabriel Limaverde-Sousa 2, Ana M. Tomas 3,4, Reviewed by: Helena Castro 3 and Elmo E. Almeida-Amaral 1* Wanderley De Souza, Federal University of Rio de Janeiro, 1 Laborato´ rio de Bioqu´ımica de Tripanosomatideos, Instituto Oswaldo Cruz (IOC), Fundac¸ão Oswaldo Cruz – FIOCRUZ, Brazil Rio de Janeiro, Brazil, 2 Laborato´ rio de Esquistossomose Experimental, Instituto Osvaldo Cruz, Fundac¸ão Oswaldo Cruz – Andrea Ilari, FIOCRUZ, Rio de Janeiro, Brazil, 3 i3S—Instituto de Investigac¸ão e Inovac¸ão em Sau´ de, Universidade do Porto, Porto, Italian National Research Portugal, 4 ICBAS—Instituto de Cieˆ ncias Biome´ dicas Abel Salazar, Universidade do Porto, Porto, Portugal Council, Italy Marina Gramiccia, ISS, Italy Leishmania infantum is a protozoan parasite that causes a vector borne infectious disease *Correspondence: in humans known as visceral leishmaniasis (VL). This pathology, also caused by L. Elmo E. Almeida-Amaral donovani, presently impacts the health of 500,000 people worldwide, and is treated elmo@ioc.fiocruz.br with outdated anti-parasitic drugs that suffer from poor treatment regimens, severe side Specialty section: effects, high cost and/or emergence of resistant parasites. In previous works we have This article was submitted to disclosed the anti-Leishmania activity of (-)-Epigallocatechin 3-O-gallate (EGCG), a Parasite and Host, flavonoid compound present in green tea leaves.
    [Show full text]
  • Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids
    RESEARCH ARTICLE Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids Diego Benítez1, Andrea Medeiros1,2, Lucía Fiestas1, Esteban A. Panozzo-Zenere3, Franziska Maiwald4, Kyriakos C. Prousis5, Marina Roussaki6, Theodora Calogeropoulou5, Anastasia Detsi6, Timo Jaeger7, Jonas Šarlauskas8, Lucíja Peterlin Mašič9, Conrad Kunick4, Guillermo R. Labadie3, Leopold Flohé2,10, Marcelo A. Comini1* 1 Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay, 2 Departamento de Bioquímica, Universidad de la República, Montevideo, Uruguay, 3 Instituto de Química Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina, 4 Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig, Braunschweig, Germany, 5 Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece, 6 Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece, 7 German Centre for Infection Research, Braunschweig, Germany, 8 Department of the Biochemistry of Xenobiotics Institute of Biochemistry, Vilnius University, Vilnius, Lithuania, 9 Department for Medicinal Chemistry, Faculty of OPEN ACCESS Pharmacy, University of Ljubljana, Ljubljana, Slovenia, 10 Department of Molecular Medicine, Università degli Studi di Padova, Padova, Italy Citation: Benítez D, Medeiros A, Fiestas L, Panozzo- Zenere
    [Show full text]
  • Tryparedoxin Peroxidase-Deficiency Commits Trypanosomes to Ferroptosis- Type Cell Death Marta Bogacz, R Luise Krauth-Siegel*
    RESEARCH ARTICLE Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis- type cell death Marta Bogacz, R Luise Krauth-Siegel* Biochemie-Zentrum der Universita¨ t Heidelberg, Heidelberg, Germany Abstract Tryparedoxin peroxidases, distant relatives of glutathione peroxidase 4 in higher eukaryotes, are responsible for the detoxification of lipid-derived hydroperoxides in African trypanosomes. The lethal phenotype of procyclic Trypanosoma brucei that lack the enzymes fulfils all criteria defining a form of regulated cell death termed ferroptosis. Viability of the parasites is preserved by a-tocopherol, ferrostatin-1, liproxstatin-1 and deferoxamine. Without protecting agent, the cells display, primarily mitochondrial, lipid peroxidation, loss of the mitochondrial membrane potential and ATP depletion. Sensors for mitochondrial oxidants and chelatable iron as well as overexpression of a mitochondrial iron-superoxide dismutase attenuate the cell death. Electron microscopy revealed mitochondrial matrix condensation and enlarged cristae. The peroxidase-deficient parasites are subject to lethal iron-induced lipid peroxidation that probably originates at the inner mitochondrial membrane. Taken together, ferroptosis is an ancient cell death program that can occur at individual subcellular membranes and is counterbalanced by evolutionary distant thiol peroxidases. DOI: https://doi.org/10.7554/eLife.37503.001 Introduction *For correspondence: Ferroptosis is characterized by the iron-dependent accumulation of cellular lipid hydroperoxides to [email protected] lethal levels. This form of regulated cell death has been implicated in the pathology of degenerative heidelberg.de diseases (e.g. Alzheimer’s, Huntington’s and Parkinson’s diseases), cancer, ischemia-reperfusion Competing interests: The injury and kidney degeneration (for recent reviews see [Doll and Conrad, 2017; Stockwell et al., authors declare that no 2017; Galluzzi et al., 2018]).
    [Show full text]
  • Trypanothione Is the Primary Target for Arsenical Drugs Against African Trypanosomes (Chemotherapy) ALAN H
    Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2607-2611, April 1989 Biochemistry Trypanothione is the primary target for arsenical drugs against African trypanosomes (chemotherapy) ALAN H. FAIRLAMB*, GRAEME B. HENDERSON, AND ANTHONY CERAMI Laboratory of Medical Biochemistry, The Rockefeller University, New York, NY 10021 Communicated by William Trager, January 3, 1989 ABSTRACT The trypanosomatid metabolite N',N5-bis- tabolite N1,N8-bis(glutathionyl)spermidine (trypanothione) (glutathionyl)spermidine (trypanothione) has been demon- (5) prompted us to investigate the interaction of this com- strated to form a stable adduct with the aromatic arsenical drug pound with aromatic arsenicals in vitro and within intact melarsen oxide [p-(4,6-diamino-s-triazinyl-2-yl)aminophenyl Trypanosoma brucei. We now report that the dithiol form of arsenoxide]. The stability constant of the melarsen-trypan- trypanothione [dihydrotrypanothione, Try(SH)2] forms a sta- othione adduct (Mel T) has been determined to be 1.05 x 107 ble adduct with melarsen oxide in vitro and in vivo and that M-1. When bloodstream Trypanosoma brucei are incubated this compound is an effective inhibitor of trypanothione with either melarsen oxide or the 2,3-dimercaptopropanol disulfide reductase, an enzyme unique to trypanosomatids. adduct of melarsen oxide (melarsoprol), Mel T is the only arsenical derivative detectable in acid-soluble extracts of the MATERIALS AND METHODS cells. Trypanothione may therefore be regarded as a primary target for aromatic arsenical derivatives against African try- All reagents were of the highest purity available. Melarsen panosomes. The selective toxic action of these compounds oxide and the adduct of melarsen oxide with 2,3-dimercap- might arise through sequestration of intracellular trypan- topropanol (Mel B) were obtained from E.
    [Show full text]
  • Substrates for the Enzyme Trypanothione Disulfide Reductase
    Proc. Nati. Acad. Sci. USA Vol. 85, pp. 5374-5378, August 1988 Biochemistry "Subversive" substrates for the enzyme trypanothione disulfide reductase: Alternative approach to chemotherapy of Chagas disease (Trypanosoma cruzi/leishmaniasis/naphthoquinone/nitrofuran) GRAEME B. HENDERSON*t, PETER ULRICH*, ALAN H. FAIRLAMBt, IAN ROSENBERG§, MIERCIO PEREIRA§, MICHAEL SELA¶1, AND ANTHONY CERAMI* *Laboratory of Medical Biochemistry, The Rockefeller University, New York, NY 10021; *Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; §Department of Medicine, New England Medical Center Hospitals, Boston, MA 02111; and $Weizmann Institute of Science, Rehovot, Israel 76100 Contributed by Michael Sela, March 2, 1988 ABSTRACT The trypanosomatid flavoprotein disulfide unusual NADPH-dependent flavoprotein disulfide reductase reductase, trypanothione reductase, is shown to catalyze one- (trypanothione reductase) (8), which maintains trypanothione electron reduction of suitably substituted naphthoquinone and in the dithiol form [Try(SH)2] within the cell. In addition, nitrofuran derivatives. A number of such compounds have trypanosomatids also possess trypanothione-dependent per- been chemically synthesized, and a structure-activity relation- oxidase activity (9, 10). Given that the antioxidant defenses of ship has been established; the enzyme is most active with trypanosomatids are based upon trypanothione, inhibition of compounds that contain basic functional groups in side-chain trypanothione reductase or subversion of its antioxidant role residues. The reduced products are readily reoxidized by within the cell represents an attractive target for the design of molecular oxygen and thus undergo classical enzyme-catalyzed drugs to treat trypanosomatid infections. redox cycling. In addition to their ability to act as substrates for Trypanothione disulfide reductase has been purified from trypanothione reductase, the compounds are also shown to Crithidia fasciculata and T.
    [Show full text]
  • Engineering the Substrate Specificity of Glutathione Reductase Toward That of Trypanothione Reduction (Trypanosomes/Molecular Modeling/Drug Design) GRAEME B
    Proc. Natl. Acad. Sci. USA Vol. 88, pp. 8769-8773, October 1991 Biochemistry Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction (trypanosomes/molecular modeling/drug design) GRAEME B. HENDERSON*t, NICHOLAS J. MURGOLO**, JOHN KURIYAN§, KLARA OSAPAY§, DOROTHEA KoMINOs¶, ALAN BERRY II, NIGEL S. SCRUTTON II, NIGEL W. HINCHLIFFEII, RICHARD N. PERHAM II, AND ANTHONY CERAMI*'** *Laboratory of Medical Biochemistry, and §Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10021; $Department of Chemistry, Rutgers University, New Brunswick, NJ 08903; and I1Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom Contributed by Anthony Cerami, June 17, 1991 ABSTRACT Glutathione reductase (EC 1.6.4.2; CAS reg- responsible for maintaining glutathione (GSSG) in its reduced istry number 9001-48-3) and trypanothione reductase (CAS state (GSH). This is an important component of the cell's registry number 102210-35-5), which are related flavoprotein defense against oxidative stress; GSH also plays a crucial disulfide oxidoreductases, have marked specificities for glu- part in the biosynthesis of the deoxyribonucleotide precur- tathione and trypanothione, respectively. A combination of sors of DNA (8). However, the trypanosomatid parasites primary sequence alignments and molecular modeling, to- responsible for African sleeping sickness, leishmaniasis, and gether with the high-resolution crystal structure of human South American Chagas disease possess, in addition to glutathione reductase, identified certain residues as potentially GSSG, a dithiol, N',N8-(bis)glutathionyl spermidine, given being responsible for substrate discrimination. Site-directed the trivial name trypanothione [T(S)2] (9) (Fig. 1). In these mutagenesis ofEscherichia coli glutathione reductase was used organisms, T(S)2 plays an important role in the maintenance to test these predictions.
    [Show full text]
  • Overexpression of Trypanosoma Rangeli Trypanothione Reductase Increases Parasite Survival Under Oxidative Stress
    1 Overexpression of Trypanosoma rangeli trypanothione reductase increases parasite survival under oxidative stress I. T. BELTRAME-BOTELHO1,2*†, P. H. STOCO1†,M.STEINDEL1,B.ANDERSSON3, E. F. PELOSO4,F.R.GADELHA4 and E. C. GRISARD1* 1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil 2 Universidade do Sul de Santa Catarina, Palhoça, SC, Brazil 3 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden 4 Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade de Campinas, Campinas, SP, Brazil (Received 25 April 2016; revised 13 September 2016; accepted 16 September 2016) SUMMARY The infectivity and virulence of pathogenic trypanosomatids are directly associated with the efficacy of their antioxidant system. Among the molecules involved in the trypanosomatid response to reactive oxygen or nitrogen species, trypa- nothione reductase (TRed) is a key enzyme. In this study, we performed a molecular and functional characterization of the TRed enzyme from Trypanosoma rangeli (TrTRed), an avirulent trypanosome of mammals. The TrTRed gene has an open reading frame (ORF) of 1473 bp (∼490 aa, 53 kDa) and occurs as a single-copy gene in the haploid genome. The predicted protein contains two oxidoreductase domains, which are equally expressed in the cytosol of epimastigotes and trypomastigotes. Nicotinamide adenine dinucleotide phosphate (NADPH) generation is reduced and endogenous H2O2 production is elevated in T. rangeli Choachí strain compared with T. cruzi Y strain epimastigotes. Oxidative stress induced by H2O2 does not induce significant alterations in TrTRed expression. Overexpression of TrTRed did not influence in vitro growth or differentiation into trypomastigotes, but mutant parasites showed increased resistance to H2O2-induced stress.
    [Show full text]
  • Roles of Trypanothione S-Transferase and Tryparedoxin Peroxidase in Resistance to Antimonials Susan Wyllie University of Dundee
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 2008 Roles of trypanothione S-transferase and tryparedoxin peroxidase in resistance to antimonials Susan Wyllie University of Dundee Tim J. Vickers Washington University School of Medicine in St. Louis Alan H. Fairlamb University of Dundee Follow this and additional works at: http://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Wyllie, Susan; Vickers, Tim J.; and Fairlamb, Alan H., ,"Roles of trypanothione S-transferase and tryparedoxin peroxidase in resistance to antimonials." Antimicrobial Agents and Chemotherapy.52,4. 1359. (2008). http://digitalcommons.wustl.edu/open_access_pubs/2369 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Roles of Trypanothione S-Transferase and Tryparedoxin Peroxidase in Resistance to Antimonials Susan Wyllie, Tim J. Vickers and Alan H. Fairlamb Antimicrob. Agents Chemother. 2008, 52(4):1359. DOI: 10.1128/AAC.01563-07. Downloaded from Published Ahead of Print 4 February 2008. Updated information and services can be found at: http://aac.asm.org/content/52/4/1359 http://aac.asm.org/ These include: REFERENCES This article cites 40 articles, 16 of which can be accessed free at: http://aac.asm.org/content/52/4/1359#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on March 8, 2014 by Washington University in St. Louis Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr.
    [Show full text]