Trypanosoma brucei and Trypanosoma cruzi Tryparedoxin Peroxidases Catalytically Detoxify Peroxynitrite via Oxidation of Fast Reacting Thiols

Abstract

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 M ؊1 s ؊1 , respectively, at pH 7.4 and 37°C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 ؋ 10 5 and 7.2 ؋ 10 5 M ؊1 s ؊1 at pH 7.4 and 37°C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn 3؉ -porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo.

A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

“…The majority of reductase activity has been attributed to 2-Cys Prx orthologs, with the exception of the bovine 1-Cys (PrxVI) [50] and human PrxV [5]. A related tryparedoxin peroxidase from the protozoan parasite Trypanosoma cruzi can also function as a PN reductase [6]. It is perhaps not surprising, therefore, that the 2-Cys Prx AsPrx-4783 would protect A. stephensi cells from PN-associated cell death.…”

“…In contrast, gene products that confer protection against RNOS have been identified, but are less well studied. The peroxiredoxins (Prxs) are a recently discovered family of antioxidant peroxidases that can reduce hydrogen peroxide and alkyl hydroperoxides to water and the corresponding alcohols, respectively, and can protect cells from widely divergent organisms against a variety of nitrosative stress challenges [2][3][4][5][6].…”

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

“…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). Two typical 2-Cys peroxiredoxins are located in the cytosol (CPX) and in the mitochondrial matrix (MPX), respectively, and efficiently scavenge H 2 O 2 , peroxynitrite, and smallchain organic hydroperoxides (11)(12)(13). Two glutathione-dependent peroxidases are located at the endoplasmic reticulum (ER) and in the cytosol, and seem to be important in the metabolization of lipidderived hydroperoxides (14).…”

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B

“…Alkylation of T. cruzi Fe-SOD thiol groups by NEM was performed by incubation of Fe-SODA or Fe-SODB (200 M) with NEM (10 mM) for 2 h in phosphate buffer (50 mM, pH 7.4) at 4°C. Excess DTT and NEM were removed immediately after incubation using HiTrap desalting columns (Amersham Biosciences) as described previously (30).…”