ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser1270

Objectives: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra-or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[01/00009-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[03/14115-2]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/52053-7]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)[480120/2007-2

Cold-Induced Changes in the Protein Ubiquitin

Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation

Determinants of Habitat Selection by Hatchling Australian Freshwater Crocodiles

Animals almost always use habitats non-randomly, but the costs and benefits of using specific habitat types remain unknown for many types of organisms. In a large lake in northwestern Australia (Lake Argyle), most hatchling (<12-month-old) freshwater crocodiles (Crocodylus johnstoni) are found in floating vegetation mats or grassy banks rather than the more widely available open banks. Mean body sizes of young crocodiles did not differ among the three habitat types. We tested four potential explanations for non-random habitat selection: proximity to nesting sites, thermal conditions, food availability, and exposure to predation. The three alternative habitat types did not differ in proximity to nesting sites, or in thermal conditions. Habitats with higher food availability harboured more hatchlings, and feeding rates (obtained by stomach-flushing of recently-captured crocodiles) were highest in such areas. Predation risk may also differ among habitats: we were twice as likely to capture a crocodile after seeing it in open-bank sites than in the other two habitat types. Thus, habitat selection of hatchling crocodiles in this system may be driven both by prey availability and by predation risk

Conserved molecular interactions in centriole-to-centrosome conversion.

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.J.F., Z.L., S.S. and N.S.D. are supported from Programme Grant to D.M.G. from Cancer Research UK. H.R. is supported from MRC Programme Grant to D.M.G. J.F. thank the British Academy and the Royal Society for Newton International Fellowship and Z.L. thanks the Federation of European Biochemical Societies for the Long-Term postdoctoral Fellowship. The authors thank Nicola Lawrence and Alex Sossick for assistance with 3D-SIM.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

Recruitment of Histone Deacetylase 3 to the Interferon-A Gene Promoters Attenuates Interferon Expression

Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection

Genetic variability and structure of jaguar (Panthera onca) in Mexican zoos

ArtículoGenealogical records of animals (studbook) are created to avoid reproduction between closely related individuals, which could cause inbreeding, particularly for such endangered species as the Panthera onca (Linnaeus, 1758). Jaguar is the largest felid in the Americas and is considered an important ecological key species. In Mexico, wild jaguar populations have been significantly reduced in recent decades, and population decline typically accompany decreases in genetic variation. There is no current census of captive jaguars in Mexico, and zoos do not follow a standardized protocol in breeding programs based on genetic studies. Here, we emphasise the importance of maintaining an adequate level of genetic variation and propose the implementation of standardised studbooks for jaguars in Mexico, mainly to avoid inbreeding. In addition, achieving the aims of studbook registration would provide a population genetic characterisation that could serve as a basis for ex situ conservation programmes

Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision