Invecchiamento cellulare e riparazione del danno ossidativo nel ciliato Euplotes raikovi: caratterizzazione dei geni specifici per le metionina-solfossido redattasi

Ciliated protozoa represent excellent material to explore the biology of ageing and rejuvenation, since they are both “complex” organisms and “simple” cells directly exposed at the environment and natural selection. Using Euplotes raikovi (a protistan whose life cycle is well characterized), I investigated the molecular resources that enable cells to repair oxidative damage of their vital macromolecules. A complex of six distinct genes that encode two forms, A and B, of methionine sulphoxide redutase (Msr, the enzyme that reduces a residue of methionine sulphoxide to methionine) was identified. The complete structure of two of these genes (designated Er-msrB and Er-msrA/B) was determined including both coding and non-coding regions; the structural analysis of the other four genes (Er-msrA1, Er-msrA2-A, Er-msrA2-B, e Er-msrA2-C) was incomplete. Er-MsrB2 (specified by one of the two open reading frames of the Er-msrA/B gene), Er-MsrB1, and Er-MsrA1 are closely related to Msr’s of other protozoa. In contrast, Er-msrA3 (specified by the second open reading frame of the Er-msrA/B gene) and Er-MsrA2 are phylogenetically related to Msr’s of prokaryotic origin. A further divergence between the Er-msr genes was detected at the functional level. Transcription of the Er-msrB gene is apparently not induced by oxidative stress, whereas transcription of the genes encoding the Msr A forms is specifically enhanced in cells subjected to oxidative stres

Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi.

Background: Deleterious phenomena of protein oxidation affect every aerobic organism and methionine residues are their elective targets. The reduction of methionine sulfoxides back to methionines is catalyzed by methionine-sulfoxide reductases (Msrs), enzymes which are particularly active in microorganisms because of their unique nature of individual cells directly exposed to environmental oxidation. Results: From the transcriptionally active somatic genome of a common free-living marine protist ciliate, Euplotes raikovi, we cloned multiple gene isoforms encoding Msr of type A (MsrA) committed to repair methionine-S-sulfoxides. One of these isoforms, in addition to including a MsrA-specific nucleotide sequence, included also a sequence specific for a Msr of type B (MsrB) committed to repair methionine-R-sulfoxides. Analyzed for its structural relationships with MsrA and MsrB coding sequences of other organisms, the coding region of this gene (named msrAB) showed much more significant relationships with Msr gene coding sequences of Rhodobacterales and Rhizobiales (Alphaproteobacteria), than of other eukaryotic organisms. Conclusions: Based on the fact that the msrAB gene is delimited by Euplotes-specific regulatory 5′ and 3′ regions and telomeric C4A4/G4T4 repeats, it was concluded that E. raikovi inherited the coding region of this gene through a phenomenon of horizontal gene transfer from species of Alphaproteobacteria with which it coexists in nature and on which it likely feeds

Polar and non-polar species of the protozoan ciliate Euplotes behave differently in response to environmental oxidative stress

Polar coastal seawaters are saturated by high oxygen concentrations, which impose very effective adaptive strategies to strive against a continuous environmental oxidative stress. We studied these strategies in Euplotes nobilii (Ciliophora: Spirotrichea), a ciliate species dwelling in Antarctic and Arctic coastal seawaters, in comparison with Euplotes raikovi, a sister species living in temperate seawaters. Cell samples of the two species were exposed to hydrogen peroxide (H2O2)-induced oxidative stress and analyzed for their survival rates and levels of expression of the genes encoding the enzyme methionine-sulfoxide reductase (Msr) A, which restores oxidized methionines (in their S form) of damaged proteins to the status of functional methionines. While 6 h of exposure to a 750-µM concentration of H2O2 did not affect E. nobilii viability, these conditions were lethal to E. raikovi. In correlation with this inter-specific difference in the cell survival to oxidative stress, the MsrA-coding genes of the two species showed different mechanisms of expression: constitutive in E. nobilii, elicited by induction in E. raikovi

Trasferimento genico orizzontale da batteri a nuclei eucariotici: un esempio nel ciliato Euplotes.

La trasmissione di geni tra organismi filogeneticamente distanti è ormai considerato un fenomeno, noto come “horizontal, or lateral gene transfer”, tra i più rilevanti nell’indirizzare l’evoluzione biologica. Sembrano esservi coinvolti tutti gli organismi, più diffusamente quelli che vivono in endosimbiosi. Ad essere trasmessi sono sia geni codificanti proteine responsabili della duplicazione genica, sia geni codificanti enzimi citoplasmatici implicati nel metabolismo cellulare. Uno di questa seconda categoria di geni è stato identificato, e denominato msrAB, nel genoma macronucleare (sinteticamente attivo) di Euplotes raikovi, in relazione a uno studio strutturale e funzionale del complesso genico che presiede la sintesi di enzimi riparatori delle reazioni ossidative cui vanno incontro le macromolecole biologiche in seguito a danni metabolici e invecchiamento cellulare. msrAB è uno dei vari geni che E. raikovi utilizza per sintetizzare le due forme, A e B, di metioninasolfossido reduttasi (Msr) deputate a ridurre i residui di metionina ossidati di una proteina di nuovo a residui non ossidati. A sostegno della sua origine proteobatterica sta il fatto che la sequenza aminoacidica MsrA che esso codifica ha un’identità dell’85% con la sequenza di MsrA di Sinorhizobium meliloti. L’integrazione strutturale e funzionale di msrAB nel genoma macronucleare di E. raikovi è invece dimostrata dal fatto che le estremità telomeriche di questo gene presentano la ripetizione dell’eptanucleotide C4A4 distintiva di tutti i mini-cromosomi (“gene-sized molecules”) che compongono il genoma macronucleare di Euplotes

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 O2 saturated environment in E. nobilii

Bicyclic [3.3.0]-Octahydrocyclopenta[<i>c</i>]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-<i>trans</i> retinol (<b>1</b>) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydro­cyclopenta­[<i>c</i>]­pyrrolo analogue <b>4</b>. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of <b>4</b>, which led to the discovery of standout antagonist <b>33</b>. Analogue <b>33</b> possesses exquisite <i>in vitro</i> RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels <i>in vivo</i> in a robust manner (>90%)