A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily.

Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature.

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The "PUFA index" -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.

Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships.

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus. The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis. The analysis revealed a significant lower number of FAD's sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.

Temporal changes in the expression of the translocator protein TSPO and the steroidogenic enzyme 5α-reductase in the dorsal spinal cord of animals with neuropathic pain: Effects of progesterone administration.

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. The presence and biological activity of steroidogenic regulatory proteins and enzymes in the spinal cord suggests that neurosteroids locally generated could modulate pain messages. In this study we explored temporal changes in the spinal expression of the 18kDa translocator protein TSPO, the steroidogenic acute regulatory protein (StAr) and the steroidogenic enzyme 5α-reductase (5α-RI/II) in an experimental model of central chronic pain. Male Sprague-Dawley rats were subjected to a SCI and sacrificed at different time points (1, 14 or 28days). The development of mechanical and cold allodynia was assessed. Injured animals showed an early increase in the mRNA levels of TSPO and 5α-RII, whereas in the chronic phase a significant decrease in the expression of 5α-RI and 5α-RII was observed, coinciding with the presence of allodynic behaviors. Furthermore, since we have shown that progesterone (PG) administration may offer a promising perspective in pain modulation, we also evaluated the expression of steroidogenic proteins and enzymes in injured animals receiving daily injections of the steroid. PG-treated did not develop allodynia and showed a marked increase in the mRNA levels of TSPO, StAR, 5α-RI and 5α-RII 28days after injury. Our results suggest that in the acute phase after SCI, the increased expression of TSPO and 5α-RII may represent a protective endogenous response against tissue injury, which is not maintained in the chronic allodynic phase. PG may favor local steroidogenesis and the production of its reduced metabolites, which could contribute to the antiallodynic effects observed after PG treatment.