Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes

Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn2+ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn2+ metal complexes with duplex DNA and RNA was studied using UV thermal and fluorescence indicator displacement assays in combination with theoretical studies. Both ligands show a high affinity for ds-DNA/RNA and selectivity for ds-RNA. The ability to interact with these duplexes is blocked upon Zn2+ coordination, which was confirmed by the low variation in the melting temperature and poor displacement of the fluorescent dye from the ds-DNA/RNA. Cell viability assays show a decrease in the cytotoxicity of the metal complexes in comparison with the free ligands, which can be associated with the observed binding to the nucleic acids

DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription

Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28–42; region 2, 66–112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription

Analysis of the Ush2a Gene in Medaka Fish (Oryzias latipes)

Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndrome is clinically classified into three types: USH1, USH2 and USH3. USH2 accounts for well over one-half of all Usher cases and mutations in the USH2A gene are responsible for the majority of USH2 cases, but also for atypical Usher syndrome and recessive non-syndromic RP. Because medaka fish (Oryzias latypes) is an attractive model organism for genetic-based studies in biomedical research, we investigated the expression and function of the USH2A ortholog in this teleost species. Ol-Ush2a encodes a protein of 5.445 aa codons, containing the same motif arrangement as the human USH2A. Ol-Ush2a is expressed during early stages of medaka fish development and persists into adulthood. Temporal Ol-Ush2a expression analysis using whole mount in situ hybridization (WMISH) on embryos at different embryonic stages showed restricted expression to otoliths and retina, suggesting that Ol-Ush2a might play a conserved role in the development and/or maintenance of retinal photoreceptors and cochlear hair cells. Knockdown of Ol-Ush2a in medaka fish caused embryonic developmental defects (small eyes and heads, otolith malformations and shortened bodies with curved tails) resulting in late embryo lethality. These embryonic defects, observed in our study and in other ciliary disorders, are associated with defective cell movement specifically implicated in left-right (LR) axis determination and planar cell polarity (PCP)

Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification

<div><p>Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly <i>Drosophila melanogaster</i> and F44G4.8/DEP-1 from the nematode <i>Caenorhabditis elegans</i>, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins.</p></div

Domain architecture of human R3 RPTP members.

<p>Schematic representation of human R3 subtype RPTP protein members. For catalytic PTP, FN3 and signal peptide symbols see the figure. Black and light blue boxes represent the transmembrane segments and the cytoplasmatic regions after the PTP domain, respectively. Note the larger size of the juxtamembrane FN3 domain in PTPRQ, PTPRB and PTPRJ proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.ref015" target="_blank">15</a>]. Protein amino acid numbers are indicated in parenthesis below the protein names.</p

Protein and DNA trees showing the evolutionary relationships between RPTPs.

<p>A) Bayes protein concatenated tree (see text for description of model parameters and analysis specifics). Values at the nodes in the tree indicate the Bayesian posterior probability for that node. B) DNA elide Bayes tree (see text for description of model parameters and analysis specifics). Values at the nodes in the tree indicate the Bayesian posterior probability for that node. Protein and DNA parsimony, and additional Bayesian phylogenetic trees are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.s006" target="_blank">S2 File</a>.</p

Invertebrate and vertebrate PTP domain similarity.

<p>Heat map representing color-coded blastp E value of PTP domains of R3 PTPRs from vertebrates and inverebrates deuterostomes, protostomes and sponges. Note the strong similarity of the sponge PTP sequence with PTPRF and PTPRG. E values are coloured from green (low similarity) to red (high similarity) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.s008" target="_blank">S1 Table</a> for numerical E values; 100% similarity corresponds to an E value of 0.0). Full scientific names of species are: Co, <i>Capsaspora owczarzaki</i>; Aq, <i>Amphimedon queenslandica</i>; Ct, <i>Capitella teleta</i>; Dm, <i>Drosophila melanogaster</i>; Ce, <i>Caenorhabditis elegans</i>; Hc, <i>Haemonchus contortus</i>; Sp, <i>Strongylocentrotus purpuratus</i>; Sk, <i>Saccoglossus kowalevskii</i>; Cs, <i>Ciona Savigny</i>; Ci, <i>Ciona intestinalis</i>; Hs, <i>Homo sapiens</i>. Other sequences Chicken (Gg) <i>Gallus gallus</i>; Fish (Dr) <i>Danio rerio</i>.</p

Correction: The Tetraspanin-Associated Uroplakins Family (UPK2/3) Is Evolutionarily Related to PTPRQ, a Phosphotyrosine Phosphatase Receptor.

[This corrects the article DOI: 10.1371/journal.pone.0170196.]