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First published online 21 April 2009
doi: 10.1242/jcs.043604

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Dominant roles of the polybasic proline motif and copper in the PrP23-89-mediated stress protection response

¹ Department of Pathology, The University of Melbourne, 3010, Australia
² Mental Health Research Institute, The University of Melbourne, 3010, Australia
³ Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3010, Australia
⁴ School of Physics, Monash University, Clayton, 3800, Australia
⁵ Centre for Neuroscience, The University of Melbourne, 3010, Australia

View larger version (29K): [in this window] [in a new window]   Fig. 1. PrP23-89 (N2) modulates intracellular oxidative stress conditional upon copper saturation. (A) Schematic representation showing the defined regions of PrP and the approximate internal cleavage site producing N2 and C2 fragments. (B-D) N2 reduces ROS induced by serum deprivation only when pre-loaded with copper. Synthetic N2 encompassing murine amino acids 23-89 was applied to serum-deprived CF10 cells in a log10 serial dilution from 0.01-10,000 nM. The 10,000 nM concentration was also applied after pre-mixing with 1-6 molar equivalents of copper; 1-6 equivalents of copper were applied without peptide for comparison. (B) Example of intracellular ROS curves obtained using the DCFDA fluorescent dye. Initial rates were calculated as the linear tangent to the curve and are shown as the percentage change from the baseline rate obtained for the serum-free environment. (C) ROS rate changes induced by the apo-PrP23-89 peptide over the dilution series. Significantly increased intracellular ROS production is seen from 0.1 nM peptide (one-way ANOVA, F=11.22, P=0.001, *P<0.01). The effect of copper-loading the PrP23-89 peptide on intracellular ROS is shown in D, with black bars indicating the copper-loaded peptide and white bars indicating the equivalent copper-alone condition. For comparison, the grey bar shows the intracellular ROS response to the peptide alone. Conditions significantly different from both the copper- and peptide-alone controls, as determined by two-way ANOVA (F=45.49, P<0.001), are indicated by *P<0.05 **P<0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Amino acids PrP23-50 alone reduce the ROS response to serum deprivation. Synthetic PrP51-89 and PrP23-50 were added to serum-deprived cells in the log10 serial dilution from 0.01-10,000 nM and the intracellular ROS response measured by DCFDA assay (A and B, black bars). PrP51-89 (10 µM) was also assayed with 1-6 molar equivalents CuCl2-6xglycine (white bars). Changes in the rate of ROS production are represented as the percentage change from the rate induced by serum deprivation alone. PrP51-89 at 0.01 nM and when loaded with 1-2 molar equivalents CuCl2-6xglycine shows no significant difference from when copper alone is applied (two-way ANOVA; F=1.225, P=0.2738). Cells treated with PrP23-50 show significantly reduced intracellular ROS in response to serum deprivation from 10-10,000 nM peptide (one-way ANOVA; F=4.774, P=0.0018, *P<0.05, **P<0.01). To eliminate the possibility of non-specific effects, a scrambled peptide (PrP23-50scram) was also assayed (B; white bars). No significant change in the rate of ROS production is seen for the PrP23-50scram peptide (one-way ANOVA, F=0.4482, P=0.8615).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Wild-type N2a cells show ROS reduction in response to PrP23-89 and PrP23-50. (A) PrP23-89 and PrP51-89 (10 µM) were added to N2a cells with and without 4 molar equivalents of CuCl2-6xglycine and assayed for the ROS produced in response to serum deprivation by the DCFDA assay. Changes in the rate of ROS production are represented as the percentage change from the rate induced by serum deprivation alone. PrP23-89 both with and without copper significantly reduced the intracellular ROS induced by serum deprivation (one-way ANOVA F=6.298, P=0.0021, *P<0.05, **P<0.01). (B) Phen green quenching experiments show that N2a cells have higher basal concentrations of copper compared with CF10 cells (Student's t-test, t=4.148, **P=0.0025), possibly explaining the ROS-reducing activity of apo PrP23-89 in these cells compared to that seen in the CF10 cells. (C) PrP23-50 (black bars) and PrP23-50scram (white bars) were added to serum-deprived cells in the log10 serial dilution from 0.01-10,000 nM. Cells treated with PrP23-50 show significantly reduced intracellular ROS in response to serum deprivation from 1000-10,000 nM peptide (one-way ANOVA, F=4.774, P=0.0018, *P<0.05, **P<0.01). No significant change in the rate of ROS production is seen for the PrP23-50scram peptide (one-way ANOVA, F=0.4482, P=0.8615).

View larger version (22K): [in this window] [in a new window]   Fig. 4. The PrP23-50 region has limited copper-binding ability, is not an antioxidant and assumes no specific secondary structure. (A) EPR spectra of PrP23-50 (i,iii) and PrP23-50scram (ii,iv) with 1 molar equivalent of Cu2+ in the absence (i,ii) and presence (iii,iv) of 2.5 molar equivalents glycine. Both peptides readily surrender their bound copper to the low-affinity chelator glycine, indicating that PrP23-50 binds copper non-specifically like any unstructured peptide, with the N-terminal amine, backbone amide(s) and water molecules being the likely ligands. The principal g|| and A|| parameters characterising the spectra (g||2.23, A (65|| Cu)170-180x10–4cm–1) are compatible with a 3N1O or 2N2O coordination sphere of equatorial ligands. Spin quantification by double-integration of the spectra indicates that PrP23-50 and PrP23-50scram bind around 1/3 and 1/2, respectively, of the observable Cu2+ bound in the presence of glycine; the unbound Cu2+ fraction forms EPR-silent copper hydroxide at pH 7 (Drew and Barnham, 2008). (B) The ability of PrP23-50 to act as an antioxidant was assessed by response to the Fenton reaction caused by H2O2 and FeSO4, with the fluorescent radical trap proxyl fluorescamine used to capture hydroxyl radicals produced. In comparison with the positive control, neither PrP23-50 nor PrP23-50scram showed any ability to reduce the radicals reaching the trap. Shown are the mean rates with s.e.m. for three independent experiments. (C) CD spectroscopy shows that both PrP23-50 and PrP23-50scram adopt a predominantly random coil structure.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Removal of cell membrane proteins or heparan sulphate, or disruption of lipid rafts, abolishes the PrP23-50-mediated intracellular ROS reduction response to serum deprivation. The effect of the cell surface environment on the protective function of PrP23-50 against intracellular ROS was assessed using the DCFDA assay. The log10 serial dilution of PrP23-50 was applied to the cells after treatment with (A) trypsin before the start of the assay, (B) 1 µM filipin III for the duration of the assay, (C) heparin lyase III at 10 mU/ml for 1 hour before the start then at 5 mU/ml for the duration of the assay, and (D) chondroitinase ABC as for the heparin lyase III treatment. For each condition the left panel shows the effect of the treatment on the production of intracellular ROS compared with serum-free media only, and the right panel shows the effect of the PrP23-50 peptide on the cells after they have been exposed to the treatment. Alterations in the rate of ROS production before PrP23-50 treatment have been compensated for in the analysis. The results of PrP23-50 treatment (in the absence of other treatment) are shown as empty grey bars in panel A for comparison. Significant results as determined by Student's t-test for changes in the rate caused by the treatment or by one-way ANOVA for changes induced by PrP23-50 are indicated by *P<0.05, **P<0.01 and ***P<0.001.

View larger version (47K): [in this window] [in a new window]   Fig. 6. The octarepeat region modulates cellular association and peptide turnover. 10 µM of the PrP23-89 fragment with and without premixing with 4 molar equivalents of CuCl2-6xglycine, and the PrP23-50 fragment were added to cells at 0 (background control) to 60 minutes. After this time cell lysates and media were harvested and western blotted to detect the fragments. (A) Example blots showing the cell-associated (upper plate) and media (lower plate) fractions compared to the original inoculums (OI). Band signals were quantified densitometrically and the signal represented as a percentage of the OI signal. Graphs show the mean and s.e.m. of (B) the cell-associated fraction, (C) the media fraction and (D) the sum of both fractions as an indicator of total loss of the peptide from the system, derived from three independent experiments. Where appropriate the single exponential rate curves for association or decay are shown (unbroken line) with the 95% CI (broken lines). Copper binding reduces the rate of cellular association of the PrP23-89 fragment and the intensity once association has reached equilibrium, but also reduces its loss from the media and the overall system. The PrP23-50 fragment shows almost no cellular association but a rapid, overall greater loss from the media and the system than the PrP23-89 fragment, even when the latter is not copper-bound.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Mutation of the proline residues 26 and 28 to alanine abolishes the activity of PrP23-50 and alters its cellular association. PrP23-50 was synthesised with proline residues 26 and 28 mutated to alanine. (A) Percentage changes in the rate of ROS production from the rate induced by serum deprivation alone as determined by DCFDA assay for the log10 serial dilution of the PrP23-50 P26/28A fragment (black bars), the PrP23-50 fragment results are shown for comparison (empty grey bars). 10 µM of the PrP23-50 P26/28A fragment was added to cells at 0 (background control) to 60 minutes. Cell lysates and media were harvested and western blotted to detect the fragment. (B) Example blots showing the cell-associated (upper plate) and media (lower plate) fractions compared to the original inoculum (OI). Band signals were quantified densitometrically and the signal represented as a percentage of the OI signal. Graphs show the single exponential rate curves for association or decay (unbroken line) with the 95% CI (broken lines) of (C) the cell-associated fraction, (D) the media fraction and (E) the sum of both fractions as an indicator of total loss of the peptide from the system, derived from three independent experiments. The PrP23-50 P26/28A fragment shows a greater propensity to associate with the cell than the PrP23-50 fragment and decays from the media and the overall system faster. Further it shows a tendency to aggregate, not seen for the PrP23-50 fragment.

View larger version (39K): [in this window] [in a new window]   Fig. 8. Mutation of the polybasic region prolines results in altered properties of PrP23-89. The PrP23-89 fragment was synthesised with prolines 26 and 28 mutated to alanine. The ability of this peptide to modulate the ROS response to serum deprivation with and without copper loading was monitored by DCFDA assay. All plots represent the mean and s.e.m. of four independent experiments, except panel F, where n=3. (A) The apo-23-89 P26/28A fragment was applied to cells in the log10 serial dilution. The ROS response compared with cells not exposed to the peptide is shown (black bars) in comparison to the PrP23-89 peptide response (empty grey bars). (B) Intracellular ROS response to serum depletion when applying 10 µM peptide with 1-6 molar equivalents CuCl2-6xglycine (black bars), compared with the response of the peptide alone (grey bar) and the response of CuCl2-6xglycine alone (white bars). Two-way ANOVA finds that the change in ROS rate at 1 and 2 equivalents copper are significantly different from the results obtained for wild-type PrP23-89 (F=14.74, P<0.0001, **P<0.01, ***P<0.001). (C) Viability of cells treated with 10 µM PrP23-89 P26/28A peptide and increasing equivalents of copper for 24 hours (circles and solid line) expressed relative to the no-copper condition, and compared with equivalent wild-type PrP23-89 (triangles and dashed line) and copper alone (squares and dotted line). Decreased viability is seen for the PrP23-89P26/28A peptide against both PrP23-89 and copper alone at 2 equivalents copper and relative to just the PrP23-89 peptide at 5 and 6 equivalents of copper (F=4.83, P=0.0422, *P<0.05, **P<0.01). (D) Cells were treated for 0 (background control) to 60 minutes with 10 µM PrP23-89 P26/28A with and without 4 molar equivalents of CuCl2-6xglycine. Cell-associated and media fractions were western blotted for the presence of the PrP23-89 P26/28A fragment compared with the original inoculum (OI). (E) Densitometric profiles measured vertically from the top to the bottom of the PrP23-89 P26/28A (solid line) and the wild-type PrP23-89 (dashed line) original inoculums. The PrP23-89 P26/28A fragment showed enhanced aggregation compared to the wild-type PrP23-89, with several dominant species appearing. (F) Densitometric quantification of the three most dominant species in the original inoculum lanes expressed as a percentage of their sum indicates copper saturation induces a shift from the dominant upper band (white bar segments) to increased dominance of the middle (pale grey bar segments) and monomeric bands (dark grey bar segments). Changes in the upper and monomeric band intensities are significant by two-way ANOVA (F=7.018, P=0.0269, *P<0.05, **P<0.01). (G) EPR spectra of (i) PrP23-89 and (ii) PrP23-89 P26/28A in the presence of 1 molar equivalent 65CuCl2 at pH 7.0. Multiple coordination modes exist in equilibrium for PrP23-89 that are very similar to those of isolated octapeptide repeat fragments at physiological pH (Drew and Barnham, 2008). These are unchanged upon mutation of prolines 26 and 28 to alanines. The co-ordination observed in PrP23-50 (Fig. 3A) does not occur here due to the higher affinity of the octarepeat copper coordination modes. (H) CD spectra show that the mutation of the proline residues does not alter secondary structure.

View larger version (41K): [in this window] [in a new window]   Fig. 9. Scheme depicting hypothetical modes of action of the N2 fragment. It is probable that the flexible N-terminus is bound to GAGs via the polybasic region even when resting at the cell membrane (A). Upon release of the N2 region by ROS the fragment is free to instigate further interactions. As N2/C2 cleavage most likely occurs when the octarepeats are already occupied to some extent with copper, new interactions might include metal-ion induced dimerisation of N2 fragments (B) or, as the octarepeats also bind lipids, coordination with GAGs and the lipid membrane environment, also transducing a protective effect via lipid signalling pathways. An alternative result of N2/C2 cleavage might be to deliver the N2 fragment to a cell-surface receptor such as the laminin receptor (Gauczynski et al., 2001), where binding to the receptor could initiate a protective signal transduction cascade (C). In the event the N2/C2 cleavage occurs when copper levels are depleted or if the fragment was outcompeted for copper by another protein, different interactions may occur with GAGs or with the lipid membrane environment, preventing the N2 fragment from interacting with the appropriate receptor and resulting an unrelated, possibly detrimental signal (D).

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