Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 Å crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation

Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Diversity of polyisoprenoids in ten Okinawan mangroves

The distribution and occurrence of polyisoprenoids (dolichols and polyprenols) in the leaves and roots of nine true Okinawan mangroves and the leaves of one associate mangrove were analyzed using two-dimensional thin layer chromatography. In the leaves, the distribution of three types (I, II, and III) of polyprenols and dolichols were detected. (I) The predominance of dolichols over polyprenols (more than 90%) was observed in Avicennia marina, Bruguiera gymnorrhiza, B. gymnorrhiza (yellow leaves), and Rhizophora stylosa. (II) The occurrence of both polyprenols and dolichols is observed in Excoecaria agallocha, Kandelia obovata, K. obovata (yellow leaves), Lumnitzera racemosa, Pemphis acidula, and Sonneratia alba. (III) The predominance of polyprenols over dolichols (more than 90%) is observed in Heritiera littoralis and Hibiscus tiliaceus. However, in the roots, a type-I distribution was observed in A. marina, B. gymnorrhiza, E. agallocha, H. littoralis and S. alba. A type-II distribution was observed in K. obovata, L. racemosa, P. acidula, and R. stylosa with no type-III distribution. The chain-length distribution of dolichols in the leaves and roots was C50–C140 and C60–C120, respectively. A similar chain-length distribution of polyprenols of C45–C140 and C65–C85 was detected in the leaves and roots respectively. Taken together, sixteen out of twenty-one tissues indicated that dolichols are more abundant than polyprenols in both leaves and roots. The present study is the first to clarify the diversity of polyisoprenoids in both the leaves and roots of mangrove, suggesting the chemotaxonomic significance of polyisoprenoids in the mangrove tree species