Homo- and hetero-oligomeric protein-protein associations explain autocrine and heterologous pheromone-cell interactions in Euplotes.

In Euplotes, protein pheromones regulate cell reproduction and mating by binding cells in autocrine or heterologous fashion, respectively. Pheromone binding sites (receptors) are identified with membrane-bound pheromone isoforms determined by the same genes specifying the soluble forms, establishing a structural equivalence in each cell type between the two twin proteins. Based on this equivalence, autocrine and heterologous pheromone/receptor interactions were investigated analyzing how native molecules of pheromones Er-1 and Er-13, distinctive of mating compatible E. raikovi cell types, associate into crystals. Er-1 and Er-13 crystals are equally formed by molecules that associate cooperatively into oligomeric chains rigorously taking a mutually opposite orientation, and each burying two interfaces. A minor interface is pheromone-specific, while a major one is common in Er-1 and Er-13 crystals. A close structural inspection of this interface suggests that it may be used by Er-1 and Er-13 to associate into heterodimers, yet inapt to further associate into higher complexes. Pheromone-molecule homo-oligomerization into chains accounts for clustering and internalization of autocrine pheromone/receptor complexes in growing cells, while the heterodimer unsuitability to oligomerize may explain why heterologous pheromone/receptor complexes fail clustering and internalization. Remaining on the cell surface, they are credited with a key role in cell-cell mating adhesion.

A new dataset of pheromone and pheromone-gene structures from the ciliate, Euplotes crassus.

Like many other organisms, ciliates communicate and interact socially via diffusible chemical signals, named pheromones, that are functionally associated with a genetic mating-type mechanism of cell self/not-self recognition. In Euplotes species, pheromones form species-specific families of small, globular, and disulfide-rich proteins folding into exclusively helical secondary structures. Each is specified by one of a series of high-multiple alleles that are inherited in Mendelian fashion with relationships of co-dominance at the so-called mat genetic locus of the cell transcriptionally inert micronuclear genome, and expressed in the transcriptionally active macronuclear genome as individual DNA molecules in which the central coding region is flanked by 5'-leader and 3'-trailer noncoding regions ending with C4A4/T4G4 telomeric repeats. In E. crassus, a cosmopolitan marine species with a long tradition in the study of ciliate mating systems and breeding patterns, oligonucleotides specific to amino acid sequences of pheromones Ec-1 and Ec-α were previously used to clone and sequence a first set of four structurally distinct macronuclear (mac) pheromone coding genes, mac-ec-α, mac-ec-1, mac-ec-2 and mac-ec-3, from two interbreeding strains, L-2D and POR-73. The use of these oligonucleotides in PCR amplifications of macronuclear DNA preparations from three other E. crassus interbreeding strains, ES10, Fava4 and MN4, has now resulted in the characterization of a second set of eight new pheromone coding genes, mac-ec-ß, mac-ec-γ, mac-ec-δ, mac-ec-ε, mac-ec-µ, mac-ec-4, mac-ec-5 and mac-ec-6. Multiple alignment between previously and newly determined pheromone-gene sequences reinforces the concept that the E. crassus pheromone-gene family includes two sub-families, which likely reflect a duplication of the micronuclear mat gene locus and represent an apomorphic trait of the E. crassus clade. Members of one sub-family (each identified with a Greek letter) show a 500-bp 5'-leader noncoding region rich in AGGA/AGGGA repetitions, and encode 56-amino acid pheromones with eight conserved Cys residues. Members of the other sub-family (each identified with an Arabic numeral) show an 800-bp 5'-leader noncoding region without AGGA/AGGGA repetitions, and encode 45-amino acid pheromones with ten conserved Cys residues.

Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells.

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-α were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-α, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F.

Primary Structure and Coding Genes of Two Pheromones from the Antarctic Psychrophilic Ciliate, Euplotes focardii.

In ciliates, diffusible cell type-specific pheromones regulate cell growth and mating phenomena acting competitively in both autocrine and heterologous fashion. In Euplotes species, these signaling molecules are represented by species-specific families of structurally homologous small, disulfide-rich proteins, each specified by one of a series of multiple alleles that are inherited without relationships of dominance at the mat-genetic locus of the germinal micronuclear genome, and expressed as individual gene-sized molecules in the somatic macronuclear genome. Here we report the 85-amino acid sequences and the full-length macronuclear nucleotide coding sequences of two pheromones, designated Ef-1 and Ef-2, isolated from the supernatant of a wild-type strain of a psychrophilic species of Euplotes, E. focardii, endemic to Antarctic coastal waters. An overall comparison of the determined E. focardii pheromone and pheromone-gene structures with their homologs from congeneric species provides an initial picture of how an evolutionary increase in the complexity of these structures accompanies Euplotes speciation.

Bioactive molecules from ciliates: Structure, activity, and applicative potential.

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions.

Crystal structure of the pheromone Er-13 from the ciliate Euplotes raikovi, with implications for a protein-protein association model in pheromone/receptor interactions.

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.

Functional chimeric genes in ciliates: An instructive case from Euplotes raikovi.

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.

The Sub-Chromosomic Macronuclear Pheromone Genes of the Ciliate Euplotes raikovi: Comparative Structural Analysis and Insights into the Mechanism of Expression.

In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear genes that are the transcriptionally active versions of codominant alleles inherited at the mating-type (mat) locus of the micronuclear genome, and encode cell type-distinctive signaling pheromones. These genes include a 225-231-bp coding region flanked by a conserved 544-bp 5'-leader region and a more variable 3'-trailer region. Two transcription initiation start sites and two polyadenylation sites associated with nonconventional signals cooperate with a splicing phenomenon of a 326-bp intron residing in the 5'-leader region in the generation of multiple transcripts from the same gene. In two of them, the synthesis of functional products depends on the reassignment to a sense codon, or readthrough of a strictly conserved leaky UAG stop codon. That this reassignment may take place is suggested by the position this codon occupies in the transcripts, close to the transcript extremity and far from the poly(A) tail. In such a case, one product is a 69-amino acid protein in search of function and the second product is a 126-amino acid protein that represents a membrane-bound pheromone isoform candidate to function as a cell type-specific binding site (receptor) of the soluble pheromones.

Molecular Structures and Coding Genes of the Water-Borne Protein Pheromones of Euplotes petzi, an Early Diverging Polar Species of Euplotes.

Euplotes is diversified into dozens of widely distributed species that produce structurally homologous families of water-borne protein pheromones governing self-/nonself-recognition phenomena. Structures of pheromones and pheromone coding genes have so far been studied from species lying in different positions of the Euplotes phylogenetic tree. We have now cloned the coding genes and determined the NMR molecular structure of four pheromones isolated from Euplotes petzi, a polar species which is phylogenetically distant from previously studied species and forms the deepest branching clade in the tree. The E. petzi pheromone genes have significantly shorter sequences than in other congeners, lack introns, and encode products of only 32 amino acids. Likewise, the three-dimensional structure of the E. petzi pheromones is markedly simpler than the three-helix up-down-up architecture previously determined in another polar species, Euplotes nobilii, and in a temperate-water species, Euplotes raikovi. Although sharing the same up-down-up architecture, it includes only two short α-helices that find their topological counterparts with the second and third helices of the E. raikovi and E. nobilii pheromones. The overall picture that emerges is that the evolution of Euplotes pheromones involves progressive increases in the gene sequence length and in the complexity of the three-dimensional molecular structure.

Revisiting fifty years of research on pheromone signaling in ciliates.

Among protists, pheromones have been identified in a great variety of algal species for their activity in driving gamete-gamete interactions for fertilization. Analogously in ciliates, pheromones have been identified for their activity in inducing the sexual phenomenon of conjugation. Although this identification was pioneered by Kimball more than fifty years ago, an effective isolation and chemical characterization of ciliate pheromones has remained confined to species of Blepharisma, Dileptus and Euplotes. In Euplotes species, in which the molecular structures have been determined, pheromones form species-specific families of structurally homologous helical, cysteine-rich, highly-stable proteins. Being structurally homologous, they can bind cells in competition with one another, raising interesting functional analogies with the families of growth factors and cytokines that regulate cell differentiation and development in higher organisms. In addition to inducing conjugation by binding cells in heterologous fashion, Euplotes pheromones act also as autocrine growth factors by binding to, and promoting the vegetative reproduction of the same cells from which they originate. This autocrine activity is most likely primary, providing a concrete example of how the original function of a molecule can be obscured during evolution by the acquisition of a new one.

Evidence for gene duplication and allelic codominance (not hierarchical dominance) at the mating-type locus of the ciliate, Euplotes crassus.

The high-multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating-type substance (pheromone). In strain L-2D, now known to be homozygous at the mating-type locus, we previously identified two pheromones (Ec-α and Ec-1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR-73 characterized by a heterozygous genotype and strong mating compatibility with L-2D strain. It was found that POR-73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec-α pheromone of L-2D cells. The other two pheromones were shown to be new and were designated Ec-2 and Ec-3 to denote their structural homology with the Ec-1 pheromone of L-2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating-type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.

Characterization and expression of the gene encoding En-MAPK1, an intestinal cell kinase (ICK)-like kinase activated by the autocrine pheromone-signaling loop in the Polar Ciliate, Euplotes nobilii.

In the protozoan ciliate Euplotes, a transduction pathway resulting in a mitogenic cell growth response is activated by autocrine receptor binding of cell type-specific, water-borne signaling protein pheromones. In Euplotes raikovi, a marine species of temperate waters, this transduction pathway was previously shown to involve the phosphorylation of a nuclear protein kinase structurally similar to the intestinal-cell and male germ cell-associated kinases described in mammals. In E. nobilii, which is phylogenetically closely related to E. raikovi but inhabits Antarctic and Arctic waters, we have now characterized a gene encoding a structurally homologous kinase. The expression of this gene requires +1 translational frameshifting and a process of intron splicing for the production of the active protein, designated En-MAPK1, which contains amino acid substitutions of potential significance for cold-adaptation.

Methionine sulfoxide reduction in ciliates: characterization of the ready-to-use methionine sulfoxide-R-reductase genes in Euplotes.

Genes encoding the enzyme methionine sulfoxide reductase type B, specific to the reduction of the oxidized methionine-R form, were characterized from the expressed (macronuclear) genome of two ecologically separate marine species of Euplotes, i.e. temperate water E. raikovi and polar water E. nobilii. Both species were found to contain a single msrB gene with a very simple structural organization encoding a protein of 127 (E. raikovi) or 126 (E. nobilii) amino acid residues that belongs to the group of zinc-containing enzymes. Both msrB genes are constitutively expressed, suggesting that the MsrB enzyme plays an essential role in repairing oxidative damages that appear to be primarily caused by physiological cell aging in E. raikovi and by interactions with an O(2) saturated environment in E. nobilii.

The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ß and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

Thermodynamic stability of psychrophilic and mesophilic pheromones of the protozoan ciliate euplotes.

Three psychrophilic protein pheromones (En-1, En-2 and En-6) from the polar ciliate, Euplotes nobilii, and six mesophilic pheromones (Er-1, Er-2, Er-10, Er-11, Er-22 and Er-23) from the temperate-water sister species, Euplotes raikovi, were studied in aqueous solution for their thermal unfolding and refolding based on the temperature dependence of their circular dichroism (CD) spectra. The three psychrophilic proteins showed thermal unfolding with mid points in the temperature range 55-70 °C. In contrast, no unfolding was observed for any of the six mesophilic proteins and their regular secondary structures were maintained up to 95 °C. Possible causes of these differences are discussed based on comparisons of the NMR structures of the nine proteins.

Coding genes and molecular structures of the diffusible signalling proteins (pheromones) of the polar ciliate, Euplotes nobilii.

In protozoan ciliates, diffusible signalling proteins (pheromones) regulate the vegetative growth and mating interactions. Here, the coding genes and the structures of the encoded pheromones were studied in genetically distinct wild-type strains representing interbreeding Antarctic and Arctic populations of the marine ciliate Euplotes nobilii. Determination of seven allelic pheromone-coding DNA sequences revealed that an unusual extension and high structural conservation of the 5' non-coding region are peculiar traits of this gene family, implying that this region is directly involved in the mechanism of pheromone gene expression, possibly through phenomena of intron splicing and/or frame-shifting. For four pheromones, the three-dimensional structures were determined by nuclear magnetic resonance spectroscopy in solution. These structures show that the pheromones represent a protein family which adapts to its polar environment by combining a structurally stable core of a three-helix bundle with extended polypeptide segments that are devoid of regular secondary structures and concomitantly show enhanced structural flexibility.

Cell aging-induced methionine oxidation causes an autocrine to paracrine shift of the pheromone activity in the protozoan ciliate, Euplotes raikovi.

Ciliates of the genus Euplotes rely on the autocrine (self) and paracrine (non-self) activities of their water-borne protein pheromones to control the two fundamental phenomena of their life cycle, i.e. vegetative (mitotic) growth and sex manifested as cell union in mating pairs. We observed that cell aging determines the synthesis of increasing concentrations of pheromones that are oxidized at the level of methionine residues which are more exposed on the molecular surface. The oxidized form of the E. raikovi pheromone Er-1 was purified and its interactions with its source cells were shown no longer to be of autocrine type directed to promote cell growth, but changed to interactions of the paracrine type directed to induce cell unions in mating pairs of the selfing type (i.e. involving genetically identical cells). These pairs generate viable offspring, like pairs formed between genetically different cells. It was therefore concluded that aging cells may paradoxically gain beneficial effects from the synthesis of oxidized forms of their pheromones. By undergoing mating in response to the interactions with these forms, they can re-initiate a new life cycle and, in fact, rejuvenate.

Isolation and structural characterization of two water-borne pheromones from Euplotes crassus, a ciliate commonly known to carry membrane-bound pheromones.

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome.

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating.

Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii, based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the three-dimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.

Molecular cold-adaptation: comparative analysis of two homologous families of psychrophilic and mesophilic signal proteins of the protozoan ciliate, Euplotes.

Unique opportunities are provided by phylogenetically closely related organisms thriving in stably cold, or temperate milieus to study adaptive modifications of structurally homologous molecules. These modifications are of keen interest in basic science as well as in biotechnology. This review highlights structural and functional specificities that differentiate two homologous families of psychrophilic and mesophilic water-borne proteins (designated as pheromones) that signal mitotic growth and sexual mating in two marine species of the protozoan ciliate Euplotes, i.e., E. nobilii, which is distributed in Antarctic and Arctic waters, and E. raikovi, which inhabits temperate waters. The two protein families show strict conservation of a common three-helix bundle in a compact core of the molecular structure, which provides long-lasting integrity and biological activity to these molecules in their natural environment. In the psychrophilic pheromone family, cold-adaptation appears to have been achieved by superimposing an integrated complex of structural modifications on this conserved scaffold. Functionally most relevant appear to be extensions of polypeptide segments devoid of regular secondary structures, a specific distribution of polar and hydrophobic amino acids, the presence of solvent-exposed clusters of negatively charged amino acid side chains, and a unique role of aromatic residues in anchoring the molecular architecture. Due to these modifications, the psychrophilic pheromones are an example of an elegant combination of high stability of the three-dimensional structures with sufficient structural plasticity for efficient functioning at their physiologically low temperatures.