Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

The unique tRNASec and its role in selenocysteine biosynthesis.

Selenium (Se) is an essential trace element for several organisms and is mostly present in proteins as L-selenocysteine (Sec or U). Sec is synthesized on its L-seryl-tRNASec to produce Sec-tRNASec molecules by a dedicated selenocysteine synthesis machinery and incorporated into selenoproteins at specified in-frame UGA codons. UGA-Sec insertion is signaled by an mRNA stem-loop structure called the SElenoCysteine Insertion Sequence (SECIS). tRNASec transcription regulation and folding have been described showing its importance to Sec biosynthesis. Here, we discuss structural aspects of Sec-tRNASec and its role in Sec biosynthesis as well as Sec incorporation into selenoproteins. Defects in the Sec biosynthesis or incorporation pathway have been correlated with pathological conditions.

The therapeutic strategies against Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate most prominently known for its role as the etiological agent of the Primary Amoebic Meningoencephalitis (PAM), a disease that afflicts the central nervous system and is fatal in more than 95% of the reported cases. Although being fatal and with potential risks for an increase in the occurrence of the pathogen in populated areas, the organism receives little public health attention. A great underestimation in the number of PAM cases reported is assumed, taking into account the difficulty in obtaining an accurate diagnosis. In this review, we summarize different techniques and methods used in the identification of the protozoan in clinical and environmental samples. Since it remains unclear whether the protozoan infection can be successfully treated with the currently available drugs, we proceed to discuss the current PAM therapeutic strategies and its effectiveness. Finally, novel compounds for potential treatments are discussed as well as research on vaccine development against PAM.

In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug.

Chagas disease, Sleeping Sickness, Nagana and Leishmaniasis are serious infections caused by protozoa of the order Kinetoplastidae. They were described over a century ago by seminal work of different physician-researchers and, despite the initial discoveries, few drugs have been made available for the treatment of these infections. The drugs available present serious efficacy and toxicity problems. Moreover, the emergence of resistant strains has rendered the development of novel chemotherapeutic strategies a priority. Auranofin is currently in use to treat rheumatoid arthritis in humans. Previous reports showed that this compound presents activity against Trypanosoma brucei and Leishmania cells. In Trypanosoma cruzi cells, auranofin resulted in a more potent compound than benznidazole in vitro when tested in different DTUs. In vivo experiments, although not decreasing T. cruzi parasitemia, decreases host mortality. Therefore, we propose auranofin as a potential alternative for a new chemotherapy in Chagas disease with the added advantage of already being approved for use in humans.

An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.

Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations.

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m3G antibodies.

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.