Causes and effects of loss of classical non-homologous end joining pathway in parasitic eukaryotes

We report frequent losses of components of the classical nonhomologous end joining pathway (C-NHEJ), one of the main eukaryotic tools for end joining repair of DNA double-strand breaks, in several lineages of parasitic protists. Moreover, we have identified a single lineage among trypanosomatid flagellates that has lost Ku70 and Ku80, the core C-NHEJ components, and accumulated numerous insertions in many protein-coding genes. We propose a correlation between these two phenomena and discuss the possible impact of the C-NHEJ loss on genome evolution and transition to the parasitic lifestyle

Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering

Catalase and Ascorbate Peroxidase in Euglenozoan Protists

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids

Functional differentiation of Sec13 paralogues in the euglenozoan protists

The β-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex. This suggests that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells, and in the vast majority of eukaryotes, a single Sec13 gene is present. Here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists, possess two Sec13 paralogues. Furthermore, based on protein interactions and localization studies we show that in diplonemids Sec13 functions are divided between the Sec13a and Sec13b paralogues. Specifically, Sec13a interacts with COPII and the NPC, while Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in euglenozoan flagellates

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code

Abstract Background Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. Results We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. Conclusions In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate

Expression of the <i>RbcS</i> and <i>rbcL</i> genes in <i>Euglena gracilis</i> and <i>Euglena longa</i>.

<p>Expression levels of <i>RbcS</i> and <i>rbcL</i> mRNAs were analyzed by quantitative RT-PCR and normalized over the 18S ribosomal RNA. Cultivation conditions and species are denoted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158790#pone.0158790.g002" target="_blank">Fig 2</a>.</p

Abundance of the RBCS and RBCL proteins in <i>Euglena gracilis</i> and <i>Euglena longa</i>.

<p>Protein immunodetection was performed using anti-RBCS, anti-RBCL, and anti-Tubulin antibodies. Three bands with different molecular weights were observed in anti-RBCS immunoblotting. The ~130 kDa band (marked *1) corresponds to polyprotein synthesized in the nucleus. The ~15 kDa band (marked *3) corresponds to the processed monomer after cleavage of the signal sequence and excision of decapeptides. The ~22 kDa band (marked *2) possibly corresponds to a monomer still attached to the transit peptide. The identity of the RBCL protein (arrowhead in the anti-RBCL panel) was confirmed by mass-spectrometry. Tubulin served as a loading control. Molecular weights in kDa are indicated on the left. EG-, <i>E</i>. <i>gracilis</i> cultivated photosynthetically (without ethanol); EG+, <i>E</i>. <i>gracilis</i> cultivated mixotrophically (with ethanol); EL, <i>E</i>. <i>longa</i>.</p

RuBisCO in Non-Photosynthetic Alga <i>Euglena longa</i>: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation

<div><p><i>Euglena longa</i>, a close relative of the photosynthetic model alga <i>Euglena gracilis</i>, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (<i>rbcL</i>). Here we provide new data illuminating the putative role of RuBisCO in <i>E</i>. <i>longa</i>. We demonstrated that the <i>E</i>. <i>longa</i> RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to <i>E</i>. <i>gracilis</i>, <i>E</i>. <i>longa</i> harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in <i>E</i>. <i>longa</i>, but their abundance is very low compared to <i>E</i>. <i>gracilis</i>. No RBCS monomers could be detected in <i>E</i>. <i>longa</i>, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown <i>E</i>. <i>gracilis</i>, but in <i>E</i>. <i>longa</i>, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in <i>E</i>. <i>longa</i> and suggest that its biological role in this species may be rather unorthodox, if any.</p></div