Academic literature on the topic 'Tryparedoxin peroxidase'

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.

ABSTRACT The clinical value of antimonial drugs, the mainstay therapy for leishmaniasis, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is required. Using the model organism Leishmania tarentolae, we have examined the role of trypanothione S-transferase (TST) in trivalent antimony [Sb(III)] resistance. TST has S-transferase activity with substrates such as chlorodinitrobenzene as well as peroxidase activity with alkyl and aryl hydroperoxides but not with hydrogen peroxide. Although S-transferase activity and TST protein levels were unchanged in Sb(III)-sensitive and -resistant lines, rates of metabolism of hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide were significantly increased. Elevated peroxidase activities were shown to be both trypanothione and tryparedoxin dependent and were associated with the overexpression of classical tryparedoxin peroxidase (TryP) in the cytosol of L. tarentolae. The role of TryP in Sb(III) resistance was verified by overexpression of the recombinant Leishmania major protein in Sb(III)-sensitive promastigotes. An approximate twofold increase in the level of TryP activity in this transgenic cell line was accompanied by a significant decrease in sensitivity to Sb(III) (twofold; P < 0.001). Overexpression of an enzymatically inactive TryP failed to result in Sb(III) resistance. This indicates that TryP-dependent resistance is not due to sequestration of Sb(III) and suggests that enhanced antioxidant defenses may well be a key feature of mechanisms of clinical resistance to antimonial drugs.

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k=7.2×105 M−1·s−1) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein–DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.

TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.

A capacidade de sobrevivência da Leishmânia no interior de células especializadas na destruição de patógenos deve-se à capacidade do parasito de burlar a propriedade microbicida pela produção de moléculas denominadas fatores de virulência. Dentre as proteínas diferencialmente expressas em um estudo prévio de nosso laboratório, encontramos isoformas da OPB, uma serino peptidase e da CPX, proteína antioxidante. De fato, promastigotas de L. (L.) major deficientes em OPB apresentaram significante redução na infecção e sobrevivência em macrófagos in vitro e lesões de evolução mais lenta no modelo murino de infecção na pata. De forma análoga, promastigotas de L. (L.) donovani superexpressoras de CPX apresentaram maior carga parasitária em macrófagos in vitro. Considerando essas informações e a importância da L. (L.) amazonensis na epidemiologia da leishmaniose no Brasil, nosso objetivo é analisar a importância da OPB e CPX na virulência desta espécie utilizando parasitas superexpressores e proteínas solúveis em modelos murinos de infecção in vitro e in vivo.
The survivability of Leishmania within specialized cells in the destruction of pathogens due to the parasite\'s ability to circumvent the microbicidal property for the production of molecules called virulence factors. Among the proteins differentially expressed in a previous study from our laboratory, we found isoforms of OPB, a peptidase serine and CPX, antioxidant protein. Indeed, promastigotes of L. (L.) Major disabled in OPB showed a significant reduction in infection and survival in macrophages in vitro and slower evolution of lesions in a murine model of infection in the leg. Similarly, promastigotes of L. (L.) Donovani overexpressors CPX showed higher parasite load in macrophages in vitro. Given this information and the importance of L. (L.) amazonensis in the epidemiology of leishmaniasis in Brazil, our goal is to analyze the importance of OPB and CPX virulence of this species using overexpressors parasites and soluble proteins in murine models of infection in vitro and in alive.

La leishmaniosi è una malattia parassitaria che ogni anno affligge più di 1.3 milioni di persone in tutto il mondo. È' principalmente diffusa nelle zone tropicali e subtropicali ed è caratterizzata da due forme principali: Viscerale e Cutanea. Entrambe sono causate da un protozoo del genere Leishmania e trasmesse per mezzo di pappataci del genere Phlebotomus e Lutzomya. Le terapie attualmente disponibili per la leishmaniosi sono basate su farmaci antimoniali, i quali sono caratterizzati sia da costi poco accessibili che da un'elevata tossicità. Inoltre, casi di farmaco-resistenza a questi trattamenti stanno via via aumentando, in particolare nei paesi dove la malattia è endemica. Quindi, c'è un urgente bisogno di nuovi farmaci che siano allo stesso tempo più economici e meno tossici. A differenza dei mammiferi, il metabolismo di Leishmania è basato su un ditiolo a basso peso molecolare, nel quale il tripanotione (N1,N8-bis(glutathionyl)spermidine), T(SH)2, tripanotione reduttasi (TR), triparedossina (TXN) e triparedossina perossidasi (TXNPx) sostituiscono i sistemi redox presenti nell'ospite mammifero. Infatti, questi fanno parte di un catena di trasporto elettronico che trasferisce equivalenti riducenti dall'NADPH all'H2O2 o altri perossidi organici prodotti dal macrofago durante l'infezione. Di conseguenza, la conoscenza della struttura e del meccanismo di azione di questi enzimi gioca un ruolo fondamentale nello sviluppo di nuovi inibitori, dal momento che questi risultano fondamentali alla sopravvivenza del parassita e assenti nell'ospite mammifero. Il lavoro riportato in questa tesi si focalizza sullo studio strutturale e funzionale di due di questi enzimi: TR e TXNPx. In particolare, esperimenti cinetici effettuati sulla tripanotione reduttasi da Leishmania infantum hanno permesso di studiare l'attività O2-ossidoreduttasica di tale enzima, che può ancora ossidare l'NADPH in assenza di tripanotione usando come accettare di elettroni ossigeno molecolare. Inoltre, saggi di inibizione hanno permesso l'identificazione di un nuovo composto, RDS777, (6-sec- Butoxy-2-[(3-chlorophenyl)sulfanyl]-4-pyrimidinamine), capace di inibire TR con alta efficienza (KI = 5.2 ± 3.8 μM). Per di più, la struttura cristallografica di TR in complesso con RDS777 è stata risolta e questa ha permesso l'identificazione dei residui chiave necessari all'interazione enzima-inibitore. In questa tesi è anche riportata la struttura cristallografica di TXNPx nella conformazione Fully Folded (FF), ottenuta ad una risoluzione maggiore (2.34 Å) rispetto alla precedente nella conformazione Locally Unfolded (LU). Attraverso questa nuova struttura, è stato possibile visualizzare tutti i residui che prendono parte al processo catalitico, non visibili nella conformazione LU. Queste informazioni strutturali sono state usate in studi di high throughput docking (HTD), allo scopo di identificare nuovi potenziali inibitori per questo enzima. I composti ottenuti tramite HTD sono stati testati in vitro attraverso esperimenti SPR, mentre la loro attività enzimatica è stata studiata attraverso saggi spettrofotometrici. Entrambi hanno permesso l'identificazione di 9 composti capaci di inibire TXNPx con una KD compresa tra 39 e 290 μM.
Leishmaniasis is a parasitic disease, which afflicts more than 1.3 million people throughout the world. It is more common in tropical and subtropical areas, where recurs by two main forms, Visceral and Cutaneous leishmaniasis, both caused by protozoan parasite of genus Leishmania and transmitted by the bite of the female sandfly. The current treatments for leishmaniasis are based on drugs, such as pentavalent antimonials, that are characterized by a high toxicity. In addition, cases of drug resistance have recently arisen in endemic countries and every year an increasing number of resistant strains is recorded. Consequently, there is a urgent need to project new, more affordable and less toxic drugs against this disease. Unlike the mammals, Leishmania has a unique thiol-based metabolism, in which the trypanothione (N1,N8-bis(glutathionyl)spermidine), T(SH)2, trypanothione reductase (TR), tryparedoxin (TXN) and tryparedoxin peroxidase (TXNPx) replace the redox systems present in the human host. In fact, they are part of an electron transport chain that transfers electrons from NADPH to H2O2 or organic peroxides produced by the host macrophages during the infection. Thus, the knowledge of the structures and mechanisms of action of these enzymes is very important for the development of new lead compounds since they are fundamental for the parasite survival and are absent in the mammalian host. The work reported in this thesis focuses on the functional and structural study of TR and TXNPx. In particular, the kinetic studies performed on trypanothione reductase from Leishmania infantum allowed the discovery of the promiscuous behavior by this protein, which can still oxidize NADPH in the absence of trypanothione using as electron acceptor the molecular oxygen. Furthermore, the inhibitory assays performed using an in-house library allowed the identification of a new compound, RDS777, (6-sec- Butoxy-2-[(3-chlorophenyl)sulfanyl]-4-pyrimidinamine) able to inhibit L.infantum TR with high efficiency (KI = 5.2 ± 3.8 μM). Moreover, I solved the X-ray structure of TR in complex with RDS777 which disclosed the mechanism of TR inhibition by that compound and allowed the identification of the key residues necessary for the binding. In addition, in this thesis is also reported the structural analysis of TXNPx from Leishmania major in Fully Folded (FF) conformation which has been solved during my PhD thesis at a resolution higher than that of TXNPx in Locally Unfolded (LU) conformation (2.34 Å). Through this structure, I was able to visualize all the residues involved in the catalysis not visible in the already solved LU TXNPx. The structural information was used for high throughput docking (HTD) studies in order to identify new potential inhibitors of this enzyme. The compounds selected by HTD were tested in vitro for the binding using the SPR technique and for its enzymatic activity using the HRP spectrophotometric assay. These studies allowed the identification of 9 compounds able to inhibit TXNPx with a KD in a range between 39 and 290 μM.