The Prion Protein N1 and N2 Cleavage Fragments Bind to Phosphatidylserine and Phosphatidic Acid; Relevance to Stress-Protection Responses

<div><p>Internal cleavage of the cellular prion protein generates two well characterised N-terminal fragments, N1 and N2. These fragments have been shown to bind to anionic phospholipids at low pH. We sought to investigate binding with other lipid moieties and queried how such interactions could be relevant to the cellular functions of these fragments. Both N1 and N2 bound phosphatidylserine (PS), as previously reported, and a further interaction with phosphatidic acid (PA) was also identified. The specificity of this interaction required the N-terminus, especially the proline motif within the basic amino acids at the N-terminus, together with the copper-binding region (unrelated to copper saturation). Previously, the fragments have been shown to be protective against cellular stresses. In the current study, serum deprivation was used to induce changes in the cellular lipid environment, including externalisation of plasma membrane PS and increased cellular levels of PA. When copper-saturated, N2 could reverse these changes, but N1 could not, suggesting that direct binding of N2 to cellular lipids may be part of the mechanism by which this peptide signals its protective response.</p></div

Lipid binding specificity is determined by regions of N1 and N2.

<p>Lipid spot blots (incubated at pH 7) of peptides corresponding to regions of N1 and N2, including residues 23–50 (<b>A</b>), residues 51–89 comprising the copper-binding region and therefore tested with and without copper saturation (<b>B</b>) and an N1 fragment lacking the residues of the copper-binding region, Δ51–89 (<b>C</b>). For 23–50 and N1Δ51–89, copper saturation was not tested as neither fragment contains the octarepeat copper-binding domain and blotting used the N-terminally targeted 8B4 antibody as SAF32 targets residues 79–92. <b>D.</b> Lipid spot blots of a mutant N2 P26/28A fragment with and without copper saturation at pH 7 detected with SAF32. <b>E.</b> Densitometric quantification of spot intensity for the domains of N1 and N2, n = 1. <b>F.</b> Densitometric quantification of spot intensity of the P26/28A mutation of N2 with and without copper saturation. Apo N2 intensities are shown for comparison of differences between the mutated and wild-type (WT) sequence, n = 3. Significance over blank control is shown in black and significant differences in detection from the wild type sequence of N2 are shown in red, *p<0.05, ***p<0.001.</p

Lipid spot blots identify N1 fragment binding to PS and PA.

<p>A. Diagram showing full length PrP and the regions comprising the N1 and N2 cleavage fragments. <b>B.</b> Schematic indicating the lipid spot arrangement on the membrane. <b>C.</b> PrP23-111 (N1) incubation with the lipid spot blots at pH 7 and pH 5 with and without pre-loading with four molar equivalents CuCl₂ followed by western blotting with SAF32 antibody (directed against amino acids 51–89). <b>D.</b> Densitometric quantification of spot intensity, n = 3, significance over blank control is shown as *p<0.05, ***p<0.001.</p

N2 binds PS and PA lipid spots.

<p><b>A.</b> Schematic indicating the lipid spot arrangement on the membrane. <b>B.</b> PrP23-89 (N2) incubation with the lipid spot blots at pH 7 and pH 5 with and without pre-loading with four molar equivalents CuCl₂ followed by western blotting with SAF32 antibody (directed against amino acids 51–89). <b>C.</b> Densitometric quantification of spot intensity, n = 3, significance over blank control and between conditions is shown as *p<0.05, **p<0.01, ***p<0.001.</p

Concentration-Dependent Dimerization of Anthraquinone Disulfonic Acid and Its Impact on Charge Storage

9,10-Anthraquinone-2,7-disulfonic acid (AQDS) is considered a benchmark active material for aqueous organic redox flow batteries. At low concentration, AQDS demonstrates two-electron transfer at near ideal electrochemical reversibility; however, at higher concentration, AQDS displays more complex behavior presumably due to the emergence of intermolecular reactions. Here, we systematically examine the electrochemical and physical properties of AQDS solutions using a suite of electrochemical, analytical, and spectroscopic techniques. Depending on the AQDS pretreatment, concentration, solution pH, and electrolyte composition, coupled chemical and electrochemical reactions lead to different charge storage capabilities. To elucidate the underlying cause of these differences, we performed various pretreatments of AQDS, examined chemical speciation by NMR, and investigated the corresponding electrochemical properties through cyclic voltammetry and bulk electrolysis. In all cases, reversible intermolecular dimerization was detected at solution concentrations greater than 10 mM. Moreover, we found that the charge state of the formed dimers was dependent on the AQDS pretreatment and the solution pH. Under acidic conditions, 1.5 electrons per molecule of AQDS were reversibly accessible, whereas under buffered mild-alkaline conditions, only one electron per molecule of AQDS was accessible. Because of insufficient proton concentration, AQDS did not cycle reversibly in unbuffered neutral electrolyte. Even when employing chemical oxidants during a chemical titration, charge storage of two electrons per molecule could not be realized. We hypothesize that adduct formation between AQDS and CO₂, along with solution pH, play important roles in the charge accessibility

N2, but not N1, reverses PS externalisation and PA increase in the absence of further membrane changes.

<p><b>A.</b> Laurdan GP of serum starved cells alone and when treated N2 (23–89), N1 (23–111), N2 with the two prolines within the N-terminal polybasic region mutated to alanines (23-89P26/28A, the octarepeat region (51–89), copper-saturated (four molar equivalents) N1, N2, 23-89P26/28A and 51–89, 23–50 and equivalent copper without peptide, measured under the same conditions used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134680#pone.0134680.g004" target="_blank">Fig 4</a>. n = 3. <b>B.</b> Annexin V magnetic separation of N1 and N2 with and without copper saturation. Filled bar indicates 10% (v/v) serum and hollow bars show conditions with 0% serum. n = 3. <b>C.</b> NBD-PS anisotropy of N2 with and without copper saturation. n = 3. <b>D.</b> Phospholipase-D activity within N1 and N2 (+/- copper), and 23–50 (no copper) serum starved cells. n = 3. <b>E.</b> Relative PA concentrations within N2 treated (+/- copper) serum starved cells. n = 3. Fig *p<0.05.</p

Serum deprivation causes changes to PS and PA in CF10 cells.

<p><b>A.</b> Laurdan GP changes following serum deprivation for 30 minutes as compared with benzyl alcohol (BA) and filipin III controls. n = 3. <b>B.</b> Live cell imaging of NBD-PS labelled CF10 cells. Image intensity is thresholds have been selected to view detail in the staining pattern and do not represent a comparison of fluorescence intensity. Scale bars = 20 μm. <b>C.</b> Fluorescence emission spectra of NBD-PS labelled cells following transfer into serum-free medium, scans were taken immediately after media replacement. <b>D.</b> Anisotropy of NBD-PS in CF10 cells with and without serum present. n = 3. <b>E.</b> Counts from magnetic separation of cells that have lost membrane asymmetry allowing them to bind PS at 5 and 15 minutes post serum withdrawal. n = 4. <b>F.</b> ROS production detected by DCF fluorescence when cells are serum-starved and with exposure to butan-1-ol to inhibit PLD activity. n = 4. <b>G.</b> Measurement of cellular phosphatidic acid concentration 30 minutes after commencing serum deprivation. n = 3. <b>H.</b> Measurement of phospholipase-D activity following 15 minutes serum starvation. n = 3. For all panels, *p<0.05, ***p<0.001.</p

Over-expression of APP isoforms in HEK cells does not alter endogenous PrP^C.

<p>(A) Representative western blot of APP and PrP^C (antibody 3F4) in HEK cells stably transfected with either the vector alone (Hyg) or one of the APP isoforms (APP₆₉₅, APP₇₅₁, APP₇₇₀), and subsequent β-actin staining to allow adjustments for equal protein loading. Approximate molecular weights (kDa) are indicated. (B) Quantification of APP and PrP^C protein levels expressed relative to Hyg control cells (dashed line). Data from 4 independent experiments. Statistical analysis by one way ANOVA with Dunnett's post test comparison to the Hyg cells, ***p<0.001, **p<0.01, n.s. not significant.</p

Unaltered PrP^C protein levels in transgenic mice over-expressing human wild type or familial AD mutant APP.

<p>(A) Western blot of APP and PrP^C (antibody 6D11) in I5 (n = 3) and J20 (n = 2) transgenic, and age-matched non-transgenic control, mouse brain homogenates, with membrane re-probing for β-actin. Approximate molecular weights (kDa) are indicated. (B) Quantification of APP and PrP^C protein levels expressed relative to the control mice (dashed line). Error bars represent ± SD. Statistical analysis by unpaired t-test, **p<0.01, n.s. not significant.</p

<i>Ctcf</i> nullizygous embryos can develop <i>ex vivo</i> to the morula/blastocyst stage.

<p><i>Ctcf</i> nullizygous embryos can develop <i>ex vivo</i> to the morula/blastocyst stage.</p