Dual actions of nitric oxide on angiogenesis: possible roles of PKC, ERK, and AP-1

Regulation of angiogenesis by nitric oxide (NO) is controversial since NO has been shown to have both pro- and anti-angiogenic effects. In this study, we examined the effect of the NO donor, S-nitro-N-acetyl-penicillamine (SNAP), on in vitro angiogenesis, and the mechanisms involved: PKC activity, ERK and c-Jun phosphorylation, and AP-1 DNA binding activity, in microvascular endothelial cells. SNAP, at 0.5-4 mM, significantly and dose-dependently inhibited angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 80%, 83%, and 63% and 73%, respectively. SNAP at concentrations > 2mM also abolished AP-1 binding activity. Lower concentrations of SNAP (0.1-0.3 mM) significantly increased angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 46%, 60%, and 61% and 180%, respectively. These findings indicate that the dual pro- and anti-angiogenic actions of NO are dose-dependent and suggest that they are mediated by PKC and ERK acting on AP-1.     

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.     

Nitric oxide inhibits mitochondrial monoamine oxidase activity and decreases outer mitochondria membrane fluidity

The recent finding that mitochondria contain a nitric oxide (NO) synthase suggests that this compound is involved in the regulation of various mitochondrial functions. Monoamine oxidase (MAO) is embedded in the outer mitochondrial membrane. NO modulates membrane fluidity. Thus, the aim of the present work was to study the effect of NO on mitochondrial MAO activity and membrane fluidity. An outer mitochondrial membrane fraction (OMMF) was obtained from rat liver. OMMF was incubated with various concentrations of S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. MAO activity and fluidity were measured by a spectrophotometric assay and by the polarization of fluorescence technique, respectively. It was found that small concentrations of SNAP (0.4-40 microM) were capable of inhibiting MAO activity but unable to decrease fluidity significantly. In contrast, larger amounts of SNAP (40-300 microM) effectively decreased membrane fluidity, but were not able to further decrease MAO activity. This information suggests that mitochondrial MAO and membrane fluidity possess different sensitivity to the effect of NO. Unfortunately, the mechanism by which NO inhibits MAO remains unknown at present. However, it seems likely that the effect of NO on MAO activity is by a direct interaction of the compound or a metabolite to the protein.     

ATP dependence of the SNARE/caveolin 1 interaction in the hippocampus

The molecular mechanisms underlying the regulation of neurotransmission has been an open question for many years. Here, we have examined an interaction between caveolin1 and SNAREs (soluble N-ethylmalemide-sensitive factor attachment protein receptor) which may contribute to the cellular mechanisms underlying changes in synaptic strength. Previously, we reported that application of 4-aminopyridine to hippocampal slices resulted in a persistent potentiation of synaptic transmission and the induction of a short-lasting and specific 40-kDa complex composed of synaptosomal associated protein of 25 kDa (SNAP25) and caveolin1. We have characterized the binding properties of these proteins and observed that in vitro caveolin1 directly associates with both SNAP25 and syntaxin. Caveolin/SNARE interactions are enhanced in the presence of ATP by a mechanism that involves phosphorylation. While caveolin has been associated with cholesterol transport, signal transduction, and transcytosis, this study provides evidence that caveolin is also a SNARE accessory protein.     

Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.     

Rapid Anterograde Axonal Transport of the Syntaxin-SNAP 25-VAMP Complex

Abstract: During the process of docking and fusion of synaptic vesicles to the presynaptic membrane, several presynaptic proteins bind sequentially to a core complex associating two proteins of the presynaptic membrane, syntaxin and SNAP 25, and a protein of synaptic vesicles, VAMP/synaptobrevin. We have immunoprecipitated this core complex after CHAPS solubilization of pure cholinergic synaptosomes of Torpedo electric organ, using anti-syntaxin or anti-VAMP immunobeads. In parallel, we studied syntaxin and VAMP, which are transported by the rapid axonal flow to the nerve endings. We found that syntaxin and VAMP accumulating at the proximal end of an electric nerve ligature were already engaged in complexes, as in synaptosomes. In unligated nerves also, significant amounts of VAMP associate with syntaxin. The possibility that these complexes form after solubilization was eliminated because added VAMP was unable to associate with syntaxin in solubilized control nerves and because similar amounts of complex were obtained after sodium dodecyl sulfate or CHAPS solubilization. Hence, syntaxin is already associated with SNAP 25 and VAMP during axonal transport, before reaching nerve endings.     

Enteroviruses Remodel Autophagic Trafficking through Regulation of Host SNARE Proteins to Promote Virus Replication and Cell Exit

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Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol‐anchored proteins in biosynthetic and recycling routes in Madin‐Darby canine kidney cells

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol‐anchored proteins (GPI‐APs) and soluble secretory proteins in Madin‐Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI‐APs in biosynthetic pathway but also for their apical recycling and basal‐to‐apical transcytosis routes. The apical distribution of the t‐SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical‐destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.      

Opsin3糖基化位点的鉴定及糖基化修饰功能北大核心CSCD

为鉴定Opsin3(OPN3)的糖基化位点,通过使用衣霉素(tunicamycin)以及N-糖酰胺酶F(PNGase F)处理发现OPN3存在糖基化修饰;通过预测以及蛋白质氨基酸位点点突变鉴定OPN3的糖基化位点;利用SNAP标签蛋白构建了SNAP-Opsin3(SNAP-OPN3)重组蛋白,使用SNAP-OPN3重组蛋白进行糖基化修饰对OPN3功能影响的研究。在表达SNAP-OPN3重组蛋白后,使用SNAP-tag的反应底物SNAP-Surface;549以及SNAP-Cell;OregonGreen;通过荧光共聚焦显微镜观察OPN3以及OPN3糖基化位点突变体是否能够成熟转运至细胞膜。结果表明,OPN3的N82位点为OPN3的糖基化位点;SNAP标签不影响OPN3功能的正常发挥;使用SNAP反应底物可以清楚地观察到糖基化位点突变的OPN3不能正常转运至细胞膜。本研究首次鉴定了OPN3的糖基化位点,并构建了SNAP-OPN3重组蛋白,发现SNAP标签并不影响OPN3的成熟转运,并利用SNAP底物验证了糖基化修饰影响OPN3蛋白的成熟转运。     

CoagVDb: a comprehensive database for coagulation factors and their associated SAPs

The current state of the art in medical genetics is to identify and classify the functional (deleterious) or non-functional (neutral) single amino acid substitutions (SAPs), also known as non-synonymous SNPs (nsSNPs). The primary goal is to elucidate the mechanisms through which functional SAPs exert their effects, and ultimately interrogating this information for association with complex phenotypes. This work focuses on coagulation factors involved in the coagulation cascade pathway which plays a vital role in the maintenance of homeostasis in the human system. We developed an integrated coagulation variation database, CoagVDb, which makes use of the biological information from various public databases such as NCBI, OMIM, UniProt, PDB and SAPs (rsIDs/variant). CoagVDb enriched with computational prediction scores classify SAPs as either deleterious or tolerated. Also, various other properties are incorporated such as amino acid composition, secondary structure elements, solvent accessibility, ordered/disordered regions, conservation, and the presence of disulfide bonds. This specialized database provides integration of various prediction scores from different computational methods along with gene, protein, and disease information. We hope our database will act as a useful reference resource for hematologists to reveal protein structure–function relationship and disease genotype–phenotype correlation.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0028-5) contains supplementary material, which is available to authorized users.