Antarctic/Arctic pheromones:Pheromone Resources

Antarctic/Arctic pheromones:

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 (Candelori et al.,2013).

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 (Geralt et al.,2013).

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 (Vallesi et al.,2012).

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 ArcticE. 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  ( Giuseppe et al.,2011).

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 threedimensional 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 (Graziano et al., 2010).

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 (Vallesi et al., 2009; Placzek, WJ et al., 2007; Pedrini et al., 2007).

Free-living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type-specific protein signals (pheromones). One of these molecules, designated En-2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA₂₀WDTCSGGTGNCGTVCCGQCF₄₀SFPVSQSCAGMADSNDCPNA₆₀. The En-2 structure appears to be characterized by an adaptive insertion of a glycine-rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule  (Alimenti et al.,2002).

The NMR structures of the homologous pheromones Er-1, Er-10, and Er-2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3-helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half-cystine residues in the individual sequences within the preserved 3-dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er-2, which is accommodated by a transition from an α-helix in Er-1 and Er-10 to a 3₁₀-helix in Er-2. The most significant structural differences are located in the C-terminal part of the proteins, which may have an important role in specific receptor recognition (Luginbühl et al.,1994).

Felici, A., Alimenti, C., Ortenzi, C., Luporin,i P .1999.Purification and initial characterization of two pheromones from the marine Antarctic ciliate, Euplotes nobilii. Ital J Zool, 66:355–360.

Graziano Di Giuseppe , Fabrizio Erra , Fernando Dini , Claudio Alimenti , Adriana Vallesi , Bill Pedrinic , Kurt Wüthrich, and Pierangelo Luporini.2010. Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone mediated cell-cell signaling and cross-mating, Molecular Ecology 10(4):1061-1067.

Pedrini, Bill., William J. Placzek, Eda Koculi,Claudio Alimenti,Antonietta LaTerza,Pierangelo Luporiniand  Kurt Wüthrich.2007.Cold-adapted in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii. J Mol Biol., 372:277–286.

Placzek, WJ, et al. 2007. Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii. IUBMB Life 59:578–585.

Vallesi, A, et al. 2009. Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii. Mar Gen 2:27–32.

Vallesi, A., Di Giuseppe, G., Dini, F., Luporini, P. 2008.Pheromone evolution in the protozoan ciliate, Euplotes: The ability to synthesize diffusibile forms is ancestral and secondarily lost. Mol Phylogenet Evol 47:439–442.

Luginbühl, P., Ottiger, M., Mronga, S., Wüthrich, K.1994.Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi. Protein Sci., 3:1537–1546.

Ortenzi, C., et al. 2000.The autocrine mitogenic loop of the ciliate Euplotes raikovi: the pheromone membrane-bound forms are the cell binding sites and potential signalling receptors of soluble pheromones. Mol Biol Cell., 11:1445–1455.

Vallesi, A., et al. 2005. Autocrine, mitogenic pheromone receptor loop of the ciliate Euplotes raikovi: pheromone-induced receptor internalization. Eukaryot Cell., 4:1221–1227.

Vallesi, A., Giuli, G., Bradshaw, R.A., Luporini, P.1995.Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature., 376:522–524.

Luporini P, Raffioni S, Concetti A, Miceli C (1986) The ciliate Euplotes raikovi heterozygous at the mat genetic locus co-releases two individual species of mating pheromone: Genetic and biochemical evidence. Proc Natl Acad Sci USA 83:2889–2893.

Vallesi, Adriana, Claudio Alimenti, Bill Pedrini, Graziano Di Giuseppe, Fernando Dini, Kurt Wüthrich, and Pierangelo Luporini.2012. "Coding genes and molecular structures of the diffusible signalling proteins (pheromones) of the polar ciliate, Euplotes nobilii." Marine genomics 8:9-13.

Geralt, Michael., Claudio Alimenti, Adriana Vallesi, Pierangelo Luporini, and Kurt Wüthrich.2013. Thermodynamic Stability of Psychrophilic and Mesophilic Pheromones of the Protozoan Ciliate Euplotes., Biology 2: 142-150.

Alimenti, C.; Vallesi, A.; Federici, S.; di Giuseppe, G.; Dini, F.; Carratore, V.; Luporini, P.Isolation and structural characterization of two water-borne pheromones from Euplotes crassus, a ciliate commonly known to carry membrane-bound pheromones. J. Eukaryot. Microbiol.,58:234-241.

Alimenti Claudio, Claudio Ortenzi,Vito Carratore,Pierangelo Luporini.2002. Structural characterization of a protein pheromone from a cold-adapted (Antarctic) single-cell eukaryote, the ciliate Euplotes nobilii.,FEBS., 514(2-3):329–332.

Luginbühl Peter, Marcel Ottiger,Siggi Mronga,Kurt WÜthrich.1994. Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi., Protein Science.,3( 9):1537–1546.

 Giuseppe, Graziano Di.,  Fabrizio Erra, Fernando Dini, Claudio Alimenti, Adriana Vallesi, Bill Pedrini, Kurt Wüthrich, and Pierangelo Luporini.2011. Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating., Proceedings of the National Academy of Sciences of the United States of America.,108(8):3181-3186.

Candelori, Annalisa; Luporini, Pierangelo; Alimenti, Claudio; Vallesi, Adriana.2013. 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. International Journal of Molecular Sciences14(4): 7457-7467.