Overview of eukaryotic phylogeny emphasising the supergroup affiliation of organisms discussed here.

<p>Each of five recognised eukaryotic supergroups is shown as a coloured triangle to indicate that it contains a great many lineages, which are under continual diversification; groups not discussed are in gray, whilst Excavata (teal), stramenopiles, alveolates, and Rhizaria (SAR, red), and Opisthokonta (purple) are shown with icons for representative organisms. All of these groups radiated rapidly following the origin of eukaryotes and evolution of the LECA. Relationships are based on recent views of the branching order but should not be considered definitive.</p

Conservation and divergence at the nuclear envelope.

<p>The major protein and nucleic acid complexes responsible for control of gene expression, nucleocytoplasmic transport, and regulation of nuclear architecture are shown. The circular nucleus diagram is divided into three colourised sectors that correspond to those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006170#ppat.1006170.g001" target="_blank">Fig 1</a>. Elements are colourised that are known to deviate from likely LECA components, whilst unknown elements are shown as open symbols. Mixed purple/green is used to designate factors that are shared between Opisthokonts and Apicomplexa. Significantly, the extensively studied <i>Homo sapiens</i> nucleus appears to retain much of the machinery of the LECA, whilst trypanosomes have several clear examples of divergent molecular systems that subtend nuclear functions. In Apicomplexa, the basic nuclear system appears once more to be similar to the LECA, although several aspects (for example, the composition of the nuclear pore complex and the identity of the lamina) remain unknown at this time; evidence suggests that Apicomplexa do not possess a LECA/mammalian type lamina, suggesting the presence of a novel machinery awaiting discovery.</p

Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in <i>Trypanosoma cruzi</i> and <i>Trypanosoma brucei</i>, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of <i>T. brucei</i>, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In <i>T. cruzi</i> insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between <i>T. brucei</i> stages and 18 in common between the two species. While K-ac proteins from <i>T. cruzi</i> are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in <i>T. brucei</i>, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival

Heme binds specifically to the catalytic domain of TcK2 and inhibits TceIF2α phosphorylation and metacyclogenesis.

<p>(A) Prediction of heme binding sites along the TcK2 sequence according to the Hemebind software [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004618#ppat.1004618.ref033" target="_blank">33</a>]. The vertical dark bars are positive hits for each amino acid residue. (B) Absorption spectra of 10 μM hemin (dashed line), GST + 10 μM hemin (dotted line) and GST-TcK2 (dotted/dashed line), and GST-TcK2 incubated with 10 μM hemin (continuous line). (C) The figure shows the relative absorbance at the indicated concentrations of hemin incubated with GST-TcK2 at 25°C. (D) SDS-PAGE of His6x-TceIF2α incubated without (−) or with (+) GST-TcK2 for 15 min with [³²P]-γ-ATP in the absence or presence of 10 μM of the indicated porphyrins. The upper gel shows the autoradiogram and at the bottom is the same gel stained with Coomassie Blue R250. Panel (E) shows the percentages of differentiated cells (metacyclics-trypomastigotes) after 96 h in the presence of the indicated porphyrins counted by direct observation of 250 cells per replicate in the fluorescence microscope after DAPI staining. The numbers are mean and standard deviation (n = 3). The asterisk indicates a significant difference (p < 0.05) based on the t-Student test.</p

TcK2 null parasites incorporate more heme than WT cells, resulting in increased H₂O₂ and ROS levels.

<p>(A) Intracellular heme levels in wild type (WT) and TcK2 null parasites growing in the absence (empty bars) or in the presence (filled bars) of 10 μM ascorbate. (B) Zn (II) mesoporphyrin IX incorporation measured in WT and TcK2 null cells in the absence (empty bars) or in the presence (filled bars) of 10 μM heme. (C) and (D) respectively indicate the total peroxidase and FeSOD activities in WT and TcK2 null parasites. (E) and (F) respectively show the H₂O₂ and ROS levels in WT and TcK2 null cells cultivated in the absence (empty bars) or presence (filled bars) of 10 μM ascorbate. In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences based on t-Student test (p < 0.01).</p

Model of the proposed actions of TcK2 in response to heme during growth and differentiation of <i>T. cruzi</i>.

<p>TcK2 is located in the membrane of endosomes and is involved in heme accumulation inside these organelles through an unknown transporter. Extracellular heme enters the cell through ABC transporters and inhibits TcK2, allowing general translation. In heme deprivation, heme is released in the cytosol, and when cytosolic levels reach low levels, TcK2 is activated leading to TceIF2α phosphorylation, decreasing general translation and allowing expression of trypomastigote specific proteins. When excess heme accumulates in the cytosol, for example in the TcK2 null parasites, FeSODs are activated and peroxidases are inhibited resulting in the production and accumulation of H₂O_2, which outcomes in the increase of ROS levels by Fenton Reaction. We propose that the Fenton Reaction occurs extracellularly as ascorbate is not internalized by <i>T. cruzi</i> and washed parasites sustains better growth.</p

Ascorbate restores TcK2 null parasite growth, differentiation and infectivity, but not intracellular proliferation.

<p>(A) Growth of wild type (WT, white losangle) and TcK2 null cells in the absence (black circle) or in the presence of 10 μM (black inverted triangle), 1 mM (black triangle) or 10 mM (black square) ascorbate, or 5 mM N-acetyl-cysteine (white circle). (B) Growth of WT (white losangel) and TcK2 null parasites in the absence of ascorbate (black circle), or after removal of 10 μM ascorbate on day 0 (white losangle). On day 6, the cells were diluted 10 fold (see arrow). (C) and (D) show, respectively, the percentage of cellular differentiation into metacyclics-trypomastigotes after 96 h and the invasion of mammalian cells by the same number of wild type (WT) and the TcK2 null parasites in the absence (empty bars) or presence of 10 μM ascorbate (filled bars). (E) Intracellular proliferation of amastigotes after infection by the wild type (WT, white losangle), TcK2 null cells previously differentiated in the absence (black circle) or in the presence of 10 μM ascorbate (black inverted triangle). In all cases, the values are mean and standard deviation of triplicates. Asterisks indicate statistically significant differences between the WT and null lines in each case based on t-Student test (* p < 0.01, ** p < 0.3).</p

TcK2 null parasites lose electron dense organelles and their contents are not restored by ascorbate.

<p>Transmission electron micrographs of wild type (WT), TcK2 null and TcK2 null parasites maintained in 10 μM ascorbate (Bars = 2 μm). Arrowheads indicate the endosomal organelles and thin arrows sections of the mitochondrion in all cases.</p

Heme promotes parasite multiplication with accumulation of electron dense endosomes and changes in TcK2 gel migration.

<p>(A) Epimastigotes were cultivated in LIT medium prepared without adding hemin (black circle), or with 10 μg (black square) or 30 μg (black triangle) of hemin per ml and counted along the indicated times. Values are mean and standard deviation (n = 3). (B) Epimastigotes were maintained for 96 h in the absence of added hemin (−Hemin), or in the presence of 30 μg/ml of hemin, and processed for immunofluorescence with mAb 5D10, or with anti-cruzipain (Cz). The images also show the DAPI staining; the merged immunofluorescences of the two antibodies, and the corresponding field marked with dotted squares in the respective DIC images (Bars = 5 μm). (C) Transmission electron micrographs of parasites maintained in LIT prepared with 30 μg/ml of hemin (+Hemin) or without hemin supplementation (−Hemin) (Bars = 2 μm). Arrowheads indicate the reservosomes filled with electron dense material and arrows indicate the same structures with less electron dense material. Western blots using anti-TcK2-KD and anti-β-tubulin antibodies of samples prepared from parasites kept in the medium without (−) or with (+) hemin supplementation (30 μg/ml) (D), or from parasites kept 6 h in LIT medium containing hemin (10 μg/ml), PBS, or PBS with 30 μg/ml of hemin (E). The gel shows biological duplicates and below are the means and standard deviation of the TcK2 ^[Up]/TcK2^[Low] (n = 3). The differences are statistically significant (p < 0.05) based on the t-Student test.</p

Functional classification of transcripts that originated from upregulated contigs.

<p>Functional classification of transcripts that originated from upregulated contigs.</p