Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages

Carnieri, Eva Gunilla Skare; Moreno, Silvia N.J.; Docampo, Roberto

doi:10.1016/0166-6851(93)90160-y

Cited by 71 publications

(40 citation statements)

References 18 publications

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“…Enzyme Assays-Trypanothione-dependent peroxidase activity was measured by monitoring NADPH oxidation (18). A standard reaction mixture (1 ml) containing 50 mM HEPES, pH 8.0, 0.5 mM EDTA, 200 M NADPH, 0.5 M trypanothione reductase, 20 M trypanothione, 2 M recombinant TcGPXI, and 1 M TcTPNI (recombinant or trypanosomederived) was incubated at 30°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

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The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

Wilkinson

Meyer

Taylor

et al. 2002

Journal of Biological Chemistry

131

102

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Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.

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Section: Methodsmentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. droperoxide metabolizing capability, a question that until recently had been open to debate (18).…”

mentioning

confidence: 99%

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

Wilkinson

Meyer

Taylor

et al. 2002

Journal of Biological Chemistry

131

102

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“…It has been shown that the parasite antioxidant systems are essential for parasite survival and establishment of the infection in the vertebrate host (4)(5)(6)(7). In contrast to most eukaryotes, T. cruzi lacks catalase and selenium-dependent glutathione peroxidases, which are enzymes capable of rapidly metabolizing high levels of H 2 O 2 (8,9). Instead, it expresses an array of complex enzyme-mediated mechanisms in which the trypanosomatid-specific thiol trypanothione ([T(SH) 2 ], N 1 ,N 8 -bisglutathionylspermidine) plays a central role in the funneling of reducing equivalents to the different peroxidase antioxidant systems (10).…”

mentioning

confidence: 99%

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H 2 O 2 (k = 2.9 × 10 7 M −1 ·s −1 ) and catalytically decomposes H 2 O 2 using Cc as the reducing substrate with higher efficiency than Asc (k cat /K m = 2.1 × 10 5 versus 3.5 × 10 4 M −1 ·s −1 , respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H 2 O 2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp 233•+ ). Mutation of Trp 233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp 233•+ , a Cys 222 -derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H 2 O 2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp 233 and Cys 222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.Trypanosoma cruzi | heme peroxidase | oxidants | virulence | kinetics

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“…With no prospect of a vaccine, the search for parasite-specific traits exploitable in terms of chemotherapy is a priority (1). It has been reported that trypanosomatids have a limited ability to deal with reactive oxygen species (2,3). In the parasite these are generated by endogenous processes and as a result of external influences such as host immune responses and drug metabolism.…”

mentioning

confidence: 99%

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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In most aerobic organisms hemoperoxidases play a major role in H 2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H 2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

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Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages

Cited by 71 publications

References 18 publications

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

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