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The protein pheromone family of the ciliate Euplotes petzi, the earliest branching species in the Euplotes phylogentic tree
Full text linkSelf/non-self recognition in ciliates relies on signaling proteins (pheromones) synthesized in association with a genetic mating-type mechanism, that regulates the cell switching between the growth (mitotic) and sexual (mating) stages of the life cycle. In Euplotes species, these pheromones are freely released into the medium from where they can be purified in relative abundance. The knowledge of their molecular structures has so far been limited to four species, namely E. raikovi, E. octocarinatus, E. nobilii and E. crassus, that occupy varied positions in the Euplotes phylogenetic tree. Most research interest has now been focused on the pheromone family of E. petzi because of a major distinctive, phylogenetic trait of this species. Together with E. sinicus, E. petzi forms the earliest branching clade in Euplotes evolution.
Four structurally distinct E. petzi pheromones have so far been structurally characterized together with their coding genes. With respect to the other known Euplotes pheromones, they show smaller dimensions (only 32 amino acids vs. up to 108 in E. octocarinatus), a higher density of disulfide bonds (four), and a folding in which molecular districts with no regular structures equal in extension districts with regular structures represented by one extended and two single-turn alpha-helices. Considering that in the other Euplotes species pheromones have structures dominated by a bundle of three regular alpha-helices, the minimal dimensions and the relatively simple architecture of E. petzi pheromones thus indicates that the structural evolution of Euplotes pheromones involves a progressive increase of architectural complexity. And the finding that the E. petzi pheromone genes are practically half in dimensions the pheromone genes of the other Euplotes species reinforced this indication
Molecular basis of self/nonself recognition in ciliate mating type systems. REPORT of the XIIIth scientific meeting of the Italian Association of Developmental and Comparative Immunobiology (IADCI), Camerino MC.
No full textLike numerous other organisms, ciliates alternate their life cycles between vegetative (mitotic) growth and sex. However, ciliate sex is unique for at least two major aspects: (i) it becomes manifest as temporary cytoplasmic fusion between cells that carry diploid sets of genes and may equally be identical or different in their genotypes, and (ii) is under the control of a genetic mechanism of mating-types which may either be only two as is the case in various species of Paramecium and Blepharisma, or multiple as is the case in various species of Tetrahymena, Euplotes, Stylonychia and the hypotrichs in general. While the mating type binary systems recall the duality of the sex of the multi-cellular organisms, the multiple systems find much closer counterparts with the self/non-self recognition mechanisms that permit animals to react immunologically against invaders, and fungi and flowering plants to decide their evolution between self-sterility and self-fertility strategies. A persevering research interest on the multiple mating type systems of different species of Euplotes lead us to obtain relevant information on the genes and the cell type-specific diffusible signal proteins (pheromones) that interplay in the control of these systems. Functionally most relevant was the finding that, in full accord with their genetic determination provided by multiple sets of single-locus genes, these Euplotes pheromones are represented by species-specific family of structurally homologous proteins which, as such, can compete with one another for binding to their cell receptors in either autocrine (self), or paracrine-like (non-self) fashion. Cells grow in response to the pheromone binding that signals self, and temporarily shift to the sexual stage in response to the pheromone binding that signals non-self
SEGNALI CHIMICI (FEROMONI) DI UN CILIATO PSICROFILO A DISTRIBUZIONE BIPOLARE, Euplotes nobilii: MODIFICAZIONI STRUTTURALI IN FUNZIONE ADATTATIVA
No full textCeppi di popolazioni interfeconde di Euplotes nobilii raccolti da vari siti costieri antartici e artici si sono dimostrati capaci di secernere costitutivamente nell’ambiente feromoni rappresentati da proteine di 52-63 aminoacidi, attive nel promuoverne la riproduzione e il fenomeno sessuale della coniugazione. Alcuni di questi feromoni sono stati purificati da ceppi sia antartici, sia artici in quantità sufficienti per potere analizzare le modificazioni strutturali che ne permettono l’attività a temperature di congelamento dell’acqua e ne giustificano il comportamento da proteine psicrofile. Pur mantenendo il motivo architettonico a tre eliche-alfa intimamente connesse da ponti disolfuro, tipico dei feromoni caratterizzati da E. raikovi che è una specie di acque temperate strettamente correlata filogeneticamente con E. nobilii, i feromoni di E. nobilii si contraddistinguono per le seguenti peculiarità: (a) maggiore estensione (principalmente a livello dell’ammino terminale) delle regioni non strutturate a scapito di quelle strutturate; (b) minor densità di ponti disolfuro rispetto al numero di residui che compongono la catena polipeptidica; (c) maggior quantità di amino acidi polari e aromatici e minore di quelli idrofobici. Il significato adattivo di questo complesso di peculiarità strutturali e chimiche è stato avvalorato da analisi di denaturazione termica in acqua ed in assenza di agenti denaturanti, che hanno rivelato una termostabilità dei feromoni di E. nobilii marcatamente minore rispetto a quelli di E. raikovi. Mentre i primi vanno incontro a denaturazione in un intervallo di temperatura compreso tra 55 °C e 70 °C, i secondi conservano le loro strutture secondarie fino a 95 °C. Queste osservazioni aprono interessanti prospettive anche in campo biotecnologico-applicativo
Genes and pheromones of Euplotes mating types.
No full textThe nearly one hundred species of Euplotes evolved high-multiple mating systems. Based on analyses of Mendelian genetics, these systems have for long been assumed to be uniformly determined by series of multiple alleles inherited at a single micronuclear mat locus. The chemical characterization in an array of Euplotes species of water-borne signaling proteins (pheromones) specified by the macronuclear (transcriptionally active) versions of the micronuclear (transcriptionally inert) mat alleles has permitted us to evolve in the study of the biology of Euplotes mating types from an approach of Mendelian genetics to an approach of molecular genetics. We have cloned and structurally characterized families of macronuclear pheromone (mat) genes from a number of species that take different positions in the Euplotes phylogenetic tree. In accord with the prediction from Mendelian genetics, it appeared that early branching species, such as E. polaris, E. raikovi and E. nobilii, inherit their mating types at a single multi-allelic mat locus. However, in disagreement with the prediction from Mendelian genetics, late branching species, such as E. crassus and E. focardii, inherit their mating types at two distinct mat loci that are likely the result of an event of gene duplication in the micronuclear genome. One locus appears to be functionally homologous with the multi-allelic mat locus of the early branching species and is thus deputed to synthesize cell type-specific pheromones. The second locus, instead, appears to be functionally divergent and deputed to synthesize pheromones that are structurally identical among restricted sets of mutually mating compatible cell types
Self/nonself discrimination in ciliated Protozoa: the molecular basis. REPORT of the VIIIth meeting of the Italian Association for Developmental and Comparative Immunobiology (IADCI), Napoli.
No full textAs also a recent review in Cell on the "Quality
Control in Self/Nonself Discrimination" points out
(Boehm T. Cell 125: 845-858, 2006), comparative
studies of the mechanisms that avoid self-mating in
more ancient eukaryotes are thought to be of key
relevance for shedding light on the control of
specificity in self/nonself discrimination, as well as
on the evolutionary emergence of the antigen
receptors in the adaptive immune system. These
studies, however, traditionally drive most attention
on the self-incompatibility of plants, self-sterility and
allo-recognition of tunicates, mating types of fungi.
Scarce or no reference at all is made to ciliates.
Nevertheless, the ciliate highly multiple mating-type
systems are providing insightful information not only
on the molecular basis of self/nonself recognition in
more ancient organisms, but also on the central
question of how new receptor/ligand pairs are
generated in complex recognition systems.
This information essentially derives from: (i) NMR and crystallographic analyses (mostly carried
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out in collaboration with the Kurt Wuthrich’s laboratory at the ETH in Zurich) of the three-dimensional structures of a set of water-born protein signals (pheromones) produced by Euplotes species (Luporini et al. Curr. Pharm. Des. 12: 3015-3024, 2006), and (ii) the determination of the splicing mechanism by which the same cell controls its own specific diffusible signal and the (autocrine) binding receptor of this signal (Vallesi et al. Eukaryot. Cell 4: 1221-1227, 2005)
Pheromone-mediated cell-cell signaling in Euplotes
No full textIn ciliates, intra-specific cell-cell signalling is functionally associated with the genetic mechanism of the mating types. Based essentially on studies on the “binary” mating-type systems of Paramecium and Blepharisma, this mechanism has traditionally been considered as a sexual mechanism, in which the “mating-type factor” of one type of cells would bind, and stimulate to mate, only cells of the second “complementary” mating type. However, this view does not appear to be supported by genetic and structural studies of families of the water-borne protein mating-type factors (pheromones) that are synthesized by species of Euplotes (i. e., E. raikovi, E. nobilii and E. crassus) characterized by mating-type systems of “high-multiple” type. These studies suggest that the pheromone sexual activity is secondary with respect to an autocrine pheromone activity, which promotes the vegetative (mitotic) growth of the same cells from which these signalling molecules are synthesized and secreted. In accord with the genetic determination of their structural specificities through multiple series of single-locus alleles, Euplotes pheromones have been shown to possess three-dimensional architectures that closely mimic one another. Therefore, they can bind their cell-membrane receptors in a competitive fashion and form either homologous, or heterologous protein-protein complexes. The homologous complexes have been shown to be responsible for signalling a cell growth response. They are internalized through endocytotic vesicles and activate a protein-kinase dependent transduction pathway. In contrast, the heterologous complexes appear destined to remain blocked on the cell surface, where they are probably involved in promoting the cell mating response
Ciliate Mating Types and Pheromones
Full text linkThe story of our knowledge of ciliate mating types and their relevant signaling molecules, originally referred to as mating type substances/factors and today usually described as pheromones (to imply a more general function as chemicals used to communicate between individuals of the same species), is inextricably bound to our understanding about ciliate sexual activity. This activity is commonly manifested as
conjugation, autogamy in ciliates, like parthenogenesis in animals, being an additional and much more sporadic alternative. Although conjugation has been described in a variety of organisms, ciliate conjugation is a unique phenomenon of cell-cell interaction and reversible union in mating pairs, that has nothing to do with phenomena of gamete-gamete interaction and irreversible union into a synkaryon. The two ciliates (designated as gamonts) which unite into a conjugal, or mating pair are de facto hermaphrodite, vegetatively reproducing cells which will generate two sexually complementary haploid gametic nuclei, one migratory (male) and one stationary (female), only as result of a meiotic process that involves their diploid micronuclei and is triggered by the cell mating union itself
Sesso e riproduzione nei protozoi ciliati: la base molecolare
No full textNei ciliati il sesso si manifesta usualmente con il fenomeno della coniugazione, che comporta l'unione temporanea tra due cellule, che finiranno per scambiarsi geni e citoplasma. Questo fenomeno è sotto il controllo di complessi di mating type (binari in alcune specie, multipli in altre), ciascuno distinto dall'altro per mezzo di molecole segnale, usualmente note come feromoni, geneticamente controllate da serie di alleli multipli che segregano ad uno stesso locus. Una numerosa famiglia di queste molecole è stata purificata in Euplotes raikoui, e ne è stata determinata la struttura chimica e studiato il meccanismo d'azione. Tutti i membri della famiglia sono rappresentati da proteine di 37-40 amminoacidi, con sequenze che possono variare in tutte le posizioni eccetto che al terminale
arnrninico, dove risiede sempre un acido aspartico, e in corrispondenza di sei posizioni sempre occupate da cisteine tra loro legate a formare tre ponti di solfuro strettamente conservati. Nonostante le differenze che possono esistere tra un feromone e l'altro a livello di sequenza amminoacidica, la conformazione tridimensionale di queste molecole è basata su un modello comune rappresentato da un fastello molto compatto di tre strutture ad a elica con assi ad andamento anti-parallelo. Questa similarità di conformazione permette ad ogni feromone di competere con altri membri della propria famiglia nelle reazioni di legame a suoi specifici recettori di membrana, e di evocare risposte biologiche diverse in
quanto contesto-dipendenti. Quando queste reazioni sono di natura autocrina, ovvero tra un feromone e la stessa cellula che lo ha sintetizzato, si osserva una promozione della riproduzione vegetativa (mitotica) della cellula; i feromoni in questo caso mimano la funzione di «fattori di crescita». Quando le reazioni sono di natura paracrina, ovvero tra un feromone ed una cellula che ne produce uno diverso, si osserva l'induzione della formazione di coppie di coniuganti e i feromoni esercitano ora una funzione di induzione del fenomeno sessuale della coniugazione
Cold-adaptation in the ciliate Euplotes: comparative analysis of two homologous families of psychrophilic and mesophilic signal proteins.
No full textUnique 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 presentation highlights structural and functional specificities that differentiate two homologous families of psychrophilic and mesophilic water-bome 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-Iasting integrity and biological activity to these molecules in
their natura I 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
Purification and initial characterization of two pheromones from the marine Antartic cilate, Euplotes nobilii
Full text linkAmong a set of wild-type strains of Euplotes nobilii, every one derived asexually from one specimen isolated from Terra Nova Bay (Ross Sea, Antarctica), two were found to be representative of different mating types mutually capable of inducing each other to form mating pairs through pheromones constitutively secreted into the extracellular environment. Pheromones of strain AC-1 were purified to homogeneity and shown to be represented by two distinct proteins, that were denoted En-1 and En-2. En-1, secreted in amounts three-fold greater than En-2, was determined to have a molecular weight of 5617 and an asparagine at the N-terminus of its amino acid sequence, while En-2 has a molecular weight of 6290 and bears an asparctic acid at its N-terminus. The fact that En-1 and En-2 are coreleased by genetically identical cells of the same strain was taken to imply that they carry a heterozygotic combination of allelic pheromone genes and that these genes are regulated by relationships of co-dominance
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