Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.     

ICAM-1 and beta2 integrin deficiency impairs fat oxidation and insulin metabolism during fasting

Intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins play critical roles in immune responses. ICAM-1 may also participate in regulation of energy balance because ICAM-1-deficient mice become obese on a high-fat diet. We show that mice deficient in these adhesion receptors are unable to respond to fasting by up-regulation of fatty acid oxidation. Normal mice, when fasted, exhibit reduced circulating neutrophil counts and increased ICAM-1 expression and neutrophil recruitment in liver. Mice lacking ICAM-1 or beta2 integrins fail to show these responses--instead they become hypoglycemic with steatotic livers. Fasting ICAM-1-deficient mice reduce insulin more slowly than wild-type mice. This produces fasting hyperinsulinemia that prevents activation of adenosine mono-phosphate (AMP)-activated protein kinase in muscles and liver, which results in decreased import of long chain fatty acids into mitochondria. Thus, we show a new role for immune cells and their adhesion receptors in regulating metabolic response to fasting.     

CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation

Signaling through the second messengers calcium and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C family members, which bind both calcium and DAG. However, an alternative pathway may involve members of the CalDAG-GEF/RasGRP protein family, which have structural features (calcium-binding EF hands and DAG-binding C1 domains) that suggest they can function in calcium and DAG signal integration. To gain insight into the signaling systems that may be regulated by CalDAG-GEF/RasGRP family members, we have focused on CalDAG-GEFI, which is expressed preferentially in the brain and blood. Through genetic ablation in the mouse, we have found that CalDAG-GEFI is crucial for signal integration in platelets. Mouse platelets that lack CalDAG-GEFI are severely compromised in integrin-dependent aggregation as a consequence of their inability to signal through CalDAG-GEFI to its target, the small GTPase Rap1. These results suggest that analogous signaling defects are likely to occur in the central nervous system when CalDAG-GEFI is absent or compromised in function.     

Molecular Determinants of Juvenile Hormone Action as Revealed by 3D QSAR Analysis in Drosophila

Background

Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH). While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive.

Methodology/Principal Findings

To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen) at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 Å or longer than 13.5 Å, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity.

Conclusions/Significance

The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.     

HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia‐like animals

Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1‐deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro‐differentiation was almost suppressed. Neuro‐differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia‐like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia‐like brains that were treated with the cannabinoid receptor‐1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co‐regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype.     

Cell surface fibronectin and oncogenic transformation

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.     

CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism

CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin alphaIIbbeta3 and to activated platelets in a beta3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an alphaIIbbeta3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.     

The lack of ADAM17 activity during embryonic development causes hemorrhage and impairs vessel formation

Background

ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development.

Methodology/Principal Findings

Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5.

Conclusions/Significance

These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function.