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The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi. We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

The glycolytic pathway in trypanosomes is unique in the sense of sequestration of most of its enzymes within peroxisome-like organelles called glycosomes [18]. Because the glycosomal membrane is impermeable to large solutes like NAD(H), the essential reoxidation of glycolytically produced intraglycosomal NADH occurs by a shuttle mechanism involving the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate by the mitochondrial Gly-3-PDH [8]. In preparation for differentiation into PCF, the SS-BSF up-regulates a subset of mitochondrial and other proteins [21]. [...]these cells are metabolically active, motile, regulate their internal pH [22], and excrete end products of glucose metabolism in ratios different than the LS-BSF and PCF cells [21]. A substrate for this reaction-acetyl-CoA-is produced by PDH, an enzymatic complex that is present and active in the BSF mitochondrion [15,25]. [...]PDH was shown to be indispensable for BSF cells but only in the absence of threonine because under these artificial conditions, PDH was the only system supplying acetyl-CoA for the essential acetate production [25]. While the proteomic data do not show any significant differences between the monomorphic and pleiomorphic strains, future work combining proteomics and metabolomics with functional genomics should be extended to the mitochondrion of trypanosomes isolated not only from blood but also from other tissues to determine whether their metabolism is tissue specific and, if so, what is/are the mechanism(s) that control(s) the changes.

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E . histolytica , although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo -inositol dehydrogenase ( myo -IDH). Peroxisomal NAD-dependent myo -IDH appeared to be a dimeric enzyme with high affinity to myo -inositol (Km 0.044 mM) and can utilize also scyllo -inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo -IDH with PTS1 are present in E . dispar , E . nutalli and E . moshkovskii but not in E . invadens , and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti . The presence of peroxisomes in E . histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Many eukaryotic genes do not follow simple vertical inheritance. Elongation factor 1α (EF-1α) and methionine adenosyl transferase (MAT) are enzymes with complicated evolutionary histories and, interestingly, the two cases have several features in common. These essential enzymes occur as two relatively divergent paralogs (EF-1α/EFL, MAT/MATX) that have patchy distributions in eukaryotic lineages that are nearly mutually exclusive. To explain such distributions, we must invoke either multiple eukaryote-to-eukaryote horizontal gene transfers (HGTs) followed by functional replacement or presence of both paralogs in the common ancestor followed by long-term coexistence and differential losses in various eukaryotic lineages. To understand the evolution of these paralogs, we have performed in vivo experiments in Trypanosoma brucei addressing the consequences of long-term coexpression and functional replacement. In the first experiment of its kind, we have demonstrated that EF-1α and MAT can be simultaneously expressed with EFL and MATX, respectively, without affecting the growth of the flagellates. After the endogenous MAT or EF-1α was downregulated by RNA interference, MATX immediately substituted for its paralog, whereas EFL was not able to substitute for EF-1α, leading to mortality. We conclude that MATX is naturally capable of evolving patchy paralog distribution via HGTs and/or long- term coexpression and differential losses. The capability of EFL to spread by HGT is lower and so the patchy distribution of EF-1α/EFL paralogs was probably shaped mainly by deep paralogy followed by long-term coexistence and differential losses.

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

Zircon ages (U-Pb, LA-ICP-MS) obtained from skarns in the Kutná Hora and Svratka units and in the Moldanubian Zone (Bohemian Massif) show significant variations between the lithotectonic units as well as among samples from individual localities. The ages fall between 310 Ma and 3.1 Ga. We suggest that such large spread is a result of both different zircon clastic grain sources and subsequent metamorphic histories. The oldest Neoarchaean to Neoproterozoic ages are interpreted as detrital zircon grains from pre-Cadomian basement. Pronounced age maxima between 600 and 520 Ma (late Neoproterozoic to early Cambrian) were found in skarn samples from the Moldanubian Zone, Svratka and Kutná Hora units. We interpret these as maximum ages of skarn precursor deposition. Apparent Cambrian-Ordovician-Silurian to early Devonian zircon ages were obtained from non-mineralized samples of the Moldanubian Zone and also in file Kutná Hora Unit. This wide temporal interval could reflect maximum ages of skarn precursors related to disintegration of abundant early Palaeozoic magmatic rocks, provided the zircons were not modified by later metamorphism or fluid circulation. The early Carboniferous ages are interpreted to result from strong Variscan HT metamorphism and subsequent circulation of post-metamorphic fluids. Zircons of this age are dominant in skarns with massive magnetite mineralization, while the non-mineralized samples preserve the older ages.

Skarns in the Svratka Unit, in the neighbouring part of the Moldanubian Zone and in the Kutná Hora Complex were studied with respect to their metamorphic evolution, major- and trace-element geochemistry, oxygen isotopic composition and zircon ages. Skarns form competent lenses and layers in metamorphosed siliciclastic rocks and preserve some early deformation structures and several equilibrium assemblages representing the products of successive metamorphic reactions. The main rock-forming minerals, garnet and clinopyroxene, are accompanied by less abundant magnetite, amphibole, plagioclase, epidote ± quartz. In the Svratka Unit the early prograde M^sub 1^, prograde/peak M^sub 2^, and retrograde M^sub 3^ metamorphic stages have been distinguished. Metamorphic conditions in skarns of the Moldanubian Zone are limited to a relatively narrow interval of amphibolite facies. The prograde and retrograde events in the Kutná Hora Complex skarns probably took place under amphibolite-facies conditions. The presence of magnetite and the increasing proportion of the andradite component in the garnet indicate locally increased oxygen fugacity. Skarn geochemistry does not show systematic differences in the skarn composition among the three units. The regional variations are exceeded by differences among samples from individual localities. The Al^sub 2^O^sub 3^/TiO^sub 2^, Al^sub 2^O^sub 3^/Zr, TiO^sub 2^/Nb ratios point to the variable proportion of the detrital material, combined in skarn protoliths with CaO and FeO, the major non-detrital components. The skarns exhibit elevated abundances of Cu, Zn, Sn and As. The Eu/Eu* ratio varies in the range of 0.5-8.6, the total REE contents vary from 8 to 345 ppm. The lowest ςREE values (< 100 ppm) occur in skarns with magnetite mineralization. The wide intervals of ςREE and Eu/Eu* values are interpreted to indicate variations in the temperature and redox conditions among layers of the same locality and at various localities. The oxygen isotope compositions of garnets, pyroxenes and amphiboles from skarns of the Svratka Unit exhibit a range of δ^sup 18^O=0.1 to 4.1 %. In situ (laser-ablation ICP-MS) U-Pb dating of zircon from one of the Svratka Unit skarn bodies yielded a wide range of ages (0.5-2.6 Ga), supporting the detrital origin of this zircon population. The skarn protoliths were probably rocks of mixed detrital-exhalative origin deposited on the sea floor. The geological position of skarns, with their structural and metamorphic record, probably reflect tectono-metamorphic evolution shared with that of their host rocks. The geochemical characteristics, including oxygen isotopic compositions and the presence of detrital zircons with a wide range of ages exclude metasomatic, and point to a sedimentary-exhalative mode of origin for the studied skarns. [PUBLICATION ABSTRACT]