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.

Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

A membrane-bound eIF2 alpha kinase located in endosomes is regulated by heme and controls differentiation and ROS levels in Trypanosoma cruzi.

Translation initiation has been described as a key step for the control of growth and differentiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the expression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas' disease, as a transmembrane protein located in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the absence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of proliferative into infective and non-proliferative forms. Parasites lacking TcK2 lose this differentiation capacity and heme is not stored in reserve organelles, remaining in the cytosol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. The augmented level of hydrogen peroxide occurs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi.

Target of rapamycin (TOR)-like 1 kinase is involved in the control of polyphosphate levels and acidocalcisome maintenance in Trypanosoma brucei.

Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G(2) phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei.

Small-subunit rRNA processome proteins are translationally regulated during differentiation of Trypanosoma cruzi.

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.