Guénola Ricard

An anaerobic mitochondrion that produces hydrogen.

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product—biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

Evolutionary Diversity of Vertebrate Small Heat Shock Proteins

All vertebrates express multiple small heat shock proteins (sHsps), which are important components of the cellular chaperoning machinery and display a spectacular diversity of functions. This ranges from remodeling the cytoskeleton and inhibiting apoptosis to serving as structural proteins in eye lens and sperm tail. Most information is available for the 10 known mammalian sHsps, formally named HspB1–B10. Only three of them (Hsp27/B1, αA-crystallin/B4, αB-crystallin/B5) have been reported from nonmammalian vertebrates, while an apparent paralog, Hsp30/B11, is found in frogs and teleost fish. To reconstruct the evolutionary diversification of the sHsps in vertebrates, we searched for additional sHsps in genome, protein, and EST databases and sequenced some avian and amphibian sHsps (HspB2, Hsp30/B11). The urochordate Ciona intestinalis was included in the search, as the outgroup of vertebrates. Orthologs of seven mammalian sHsps were now found in other vertebrate classes. Two novel sHsps, named HspB11 and HspB12, were recognized in birds, and four novel sHsps, named HspB12–B15, in teleost fish. Secondary structure predictions of orthologous sHsps from different vertebrate classes indicate conservation of the β-sandwich structure of the functionally important C-terminal “α-crystallin domain,” while the N-terminal domains generally have α-helical structures, despite their pronounced sequence variation. The constructed chordate sHsp tree is supported by shared introns, indels, and diagnostic sequences. The tree distinguishes putative orthologous and paralogous relationships, which will facilitate the functional and structural comparison of the various vertebrate sHsps. The 15 recognized paralogous vertebrate sHsps reflect the period of extensive gene duplications early in vertebrate evolution. Eleven of these sHsps are grouped in a clade that might be specific for chordates. It is inferred that at least 13 intron insertions have occurred during the evolution of chordate sHsp genes, while a single ancient intron is maintained in some lineages, in line with the general trend of massive intron gain before or during early vertebrate radiation. Interesting is the occurrence of several head-to-head located pairs of chordate sHsp genes.

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end‐products of a prokaryotic‐type mixed‐acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome‐bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end‐products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

The Organellar Genome and Metabolic Potential of the Hydrogen-Producing Mitochondrion of Nyctotherus ovalis

It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor. However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III, IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing mitochondria.

Macronuclear genome structure of the ciliate Nyctotherus ovalis : Single-gene chromosomes and tiny introns

BackgroundNyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement.ResultsWe examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242) and cDNAs (5,484) and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21–29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway.ConclusionThe combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

BackgroundThe hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I.ResultsThe [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome.ConclusionThe hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.

Identification of Nipsnap1 as a novel auxiliary protein inhibiting TRPV6 activity

The transient receptor potential vanilloid channels 5 and 6 (TRPV5/6) are the most Ca2+-selective channels within the TRP superfamily of ion channels. These epithelial Ca2+ channels are regulated at different intra- and extracellular sites by the feedback response of Ca2+ itself, calciotropic hormones, and by TRPV5/6-associated proteins. In the present study, bioinformatics was used to search for novel TRPV5/6-associated genes. By including pull-down assays and functional analysis, Nipsnap1—a hitherto functionally uncharacterized globular protein—was identified as a novel factor involved in the regulation of TRPV6. Electrophysiological recordings revealed that Nipsnap1 abolishes TRPV6 currents. Subsequent biotinylation assays showed that TRPV6 plasma membrane expression did not change in the presence of Nipsnap1, suggesting that TRPV6 inhibition by Nipsnap1 is independently regulated from reduced cell surface channel expression. In addition, semi-quantitative reverse transcriptase PCR and immunohistochemical labeling of Nipsnap1 indicated that Nipsnap1 is expressed in mouse intestinal tissues—where TRPV6 is predominantly expressed—but that it does not co-localize with TRPV5 in the kidney. In conclusion, this study presents the first physiological function of Nipsnap1 as an associated protein inhibiting TRPV6 activity that possibly exerts its effect directly at the plasma membrane.