Omega‑3 Fatty Acids Regulate the Interaction of the Alzheimer’s Aβ(25–35) Peptide with Lipid Membranes

Polyunsaturated omega-3 fatty acids are increasingly proposed as dietary supplements able to reduce the risk of development or progression of the Alzheimer’s disease (AD). To date, the molecular mechanism through which these lipids act has not been yet univocally identified. In this work, we investigate whether omega-3 fatty acids could interfere with the fate of the Alzheimer-related amyloid peptide by tuning the microstructural and dynamical properties of the neuronal membrane. To this aim, the influence of the omega-3 lipid, 1,2-didocosahexaenoyl-<i>sn</i>-glycero-3-phosphocholine [22:6­(<i><i>cis</i></i>)­PC] on the biophysical properties of lipid bilayers, and on their interaction with the amyloid peptide fragment Aβ(25–35) has been investigated by Electron Spin Resonance (ESR), using spin-labeled phospholipids. The results show that the peptide selectively interacts with bilayers enriched in cholesterol (Chol) and sphingomyelin (SM). [22:6­(<i><i>cis</i></i>)­PC] enhances the Aβ(25–35)/membrane interaction, favoring a deeper internalization of the peptide among the lipid acyl chains and, consequently, hindering its pathogenic self-aggregation

Designed Glucopeptides Mimetics of Myelin Protein Epitopes As Synthetic Probes for the Detection of Autoantibodies, Biomarkers of Multiple Sclerosis

We previously reported that CSF114­(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114­(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114­(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients’ sera, and was able to detect higher antibody titers as compared to CSF114­(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients’ sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region

Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene

<div><p>Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR₁ in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR₁ was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR₁ over-expression compared to Met-5A cell line. Thrombin and PAR₁ activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR₁ signaling through G_q and G_12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR₁ signaling through G_i proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR₁ expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR₁ signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR₁ in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.</p></div

Neither β-catenin rescue nor deletion modify cell surface PAR₁ expression.

<p>NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR₁ expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR₁ expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).</p

NCI-H28 and REN cells express significant less amount of PAR₁ on plasma membrane.

<p>Cell surface PAR₁ expression was measured by ELISA using a polyclonal antibody which recognizes the N-terminus of PAR₁<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111550#pone.0111550-Paing1" target="_blank">[31]</a>. Antibody binding to fixed cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR₁ expression between Ctrl (Met-5A cells) and MPM (NCI-H28 and REN) cellswere significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test.</p

Cellular distribution of caveolin-1 and PAR₁ in Met-5A and NCI-H28 cells.

<p>Immunolabeling of β-catenin, caveolin-1 and PAR₁ in Met-5A and NCI-H28 cells was performed as described in Materials and Methods. The images shown are representative of many cells examined in two independent experiments. The arrows point out intracellular or plasma membrane localization of immunostained proteins. Scale Bar: 10 µm.</p

NCI-H28 cells over-express PAR₁.

<p>A, relative expression levels of PAR₁ mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR₁ protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR₁ expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.</p

PAR₁ agonist-induced G_q but not G_i signaling is impaired in NCI-H28 cells.

<p>A, thrombin-induced intracellular Ca²⁺ mobilization in HMEC-1, Met-5A, and NCI-H28 cells. B, selective-PAR1-AP-induced intracellular Ca²⁺ mobilization in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were loaded with Fluo-3AM to measure [Ca²⁺]_i variations as indicated by changes in fluorescence intensity. Fluorescence was recorded before agonist addition (F₀) and then every 3 seconds after thrombin (10 nM) or PAR₁-AP (10 µM) addition for another 120 seconds. Data shown are mean ± SEM of a single experiment done in triplicate. Experiments were repeated two additional times with similar results. The results are reported as relative fluorescence (RF = F/F₀ where F₀ is basal fluorescence and F is fluorescence recorded after cell stimulation with the agonist). C, inhibition of isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells by different concentrations of thrombin in the presence and absence of 100 nM SCH 79797. D, no effect of the selective PAR₁-AP on isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were exposed to different agonist concentrations. Assays were initiated by the addition of 1 µM isoproterenol. Production of cAMP was measured using a competition binding assay which includes the bovine adrenal cAMP binding protein and [³H]cAMP. Data shown are mean ± SEM of three independent experiments performed in triplicate. The differences between thrombin- and thrombin plus SCH 79797-treated cells were significant (**P≤0.01, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).</p

Double immunofluorescence labelling of caveolin-1 and PAR₁ in Met-5A and NCI-H28 cells.

<p>Double labelling was performed by incubating antibodies as follow: anti-PAR₁ and anti-caveolin-1; anti-PAR₁ and rabbit polyclonal anti-β-catenin; mouse monoclonal anti-β-catenin and anti-caveolin-1. To visualize double fluorescent staining, cells were incubated with Alexa Fluor 488- and Alexa Fluor 568-labeled goat anti-mouse and anti-rabbit antibodies as described in Materials and Methods. The images shown are representative of many cells examined in two independent experiments. The yellow stain indicates protein proximity (see arrows). All images were analyzed using the ImageJ program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111550#pone.0111550-Abramoff1" target="_blank">[34]</a>. PCC values are expressed as mean ± SEM of six examined area. PCC value for PAR₁/caveolin-1 colocalization was 0.77±0.05 and 0.84±0.03 in Met-5A and NCI-H28 cells, respectively. PCC values for PAR₁/β-catenin and caveolin-1/β-catenin colocalization in Met-5A cells were 0.70±0.02 and 0.55±0.04, respectively. Scale Bar: 10 µm.</p

Thrombin differently induces ERK1/2 activation in Met-5A and NCI-H28 cells.

<p>A, relative intensity of pERK1/2 immunoreactive bands quantified by densitometric scanning. Serum and growth factor starved Met-5A and NCI-H28 cells were incubated in the presence and absence of various thrombin concentrations ranging from 0.01 to 100 nM for 5 min. ERK1/2 activation was then determined using a specific anti-phospho-ERK1/2 antibody. Nitrocellulose membranes were then stripped and reprobed for total ERK1/2. Data (mean ± SEM) are expressed as fold-increase over Ctrl and are the averages of three independent experiments performed in duplicate. The differences in phosphorylated ERK1/2 level between Ctrl (vehicle treated Met-5A or NCI-H28 cells) and thrombin-treated cells were significant (*P≤0.05, **P≤0.01) by one-way ANOVA followed by Bonferroni’s multiple comparison test. B, a representative immunoblot.</p