Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.     

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.     

Preparation and characterisation of silicone-based coatings filled with carbon nanotubes and natural sepiolite and their application as marine fouling-release coatings

This article reports on the preparation and partial characterisation of silicone-based coatings filled with low levels of either synthetic multiwall carbon nanotubes (MWCNTs) or natural sepiolite (NS). The antifouling and fouling-release properties of these coatings were explored through laboratory assays involving representative soft-fouling (Ulva) and hard-fouling (Balanus) organisms. The bulk mechanical properties of the coatings appeared unchanged by the addition of low amounts of filler, in contrast to the surface properties, which were modified on exposure to water. The release of Ulva sporelings (young plants) was improved by the addition of low amounts of both NS and MWCNTs. The most profound effect recorded was the significant reduction of adhesion strength of adult barnacles growing on a silicone elastomer containing a small amount (0.05%) of MWCNTs. All the data indicate that independent of the bulk properties, the surface properties affect settlement, and more particularly, the fouling-release behaviour, of the filled materials.     

Selenium Status in Patients with Turner Syndrome: a Biochemical Assessment Related with Body Composition

Studies about selenium status in patients with Turner syndrome (TS) are non-existent in the literature. The aim of this study was to evaluate selenium status in patients with TS, while considering the different ages of the studied population and the relation with body composition. In total, 33 patients with TS were evaluated and grouped according to their developmental stages (children, adolescents, and adults). Selenium concentrations in their plasma, erythrocytes, urine, and nails were determined by using hydride generation atomic absorption spectrometry and erythrocyte glutathione peroxidase activity were measured by using Randox commercial kits. Additionally, height, weight, body fat percentage, waist circumference, and waist-height ratio were measured to characterize the patients. No differences in the selenium concentrations in the plasma, erythrocyte, urine, and nails or in the glutathione peroxidase activity were observed among the age groups (p > 0.05). The evaluated selenium levels were less than the established normal ones. The patients with larger waist circumference, body fat percentage, body mass index, and waist-height ratio showed lower glutathione peroxidase enzyme activity (p = 0.023). The present study shows that most patients with TS are deficient in selenium and that those with a greater accumulation of body fat have a lower GPx activity.     

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.     

N 6-Substituted AMPs Inhibit Mammalian Deoxynucleotide N-Hydrolase DNPH1

The gene dnph1 (or rcl) encodes a hydrolase that cleaves the 2’-deoxyribonucleoside 5’-monophosphate (dNMP) N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N ⁶-substituted AMPs, and among them, cytotoxic cytokinin riboside 5’-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.