Identification of Proteins Responsible for the Neuroprotective Effect of the Secretome Derived from Blood Cells of Remote Ischaemic Conditioned Rats

We have recently shown that the blood cell-derived secretome of remote ischaemic (RIC)-conditioned individuals provides an external source of neuroprotection. In this study, we identified the bioactive compounds from the total proteins released by those cells. Our main strategy was to separate protein–protein complexes while maintaining their native structure and testing their bioactive properties. Subsequently, we identified up- and downregulated bioactive proteins. We uncovered two bioactive fractions composed of 18 proteins. Most of the protein peaks were unchanged; however, RIC mediated a decrease in two peaks (comprising seven proteins) and an increase in one peak (identified as haptoglobin). When focussing on the biological activity of these proteins, we found positive impacts on the regulation of cellular metabolic processes and an increase in biological processes related to the acute phase response and inflammation in the RIC-treated samples. Although we have identified the 18 proteins that exert the greatest cytoprotection, additional studies are needed to elucidate their particular function and detailed mechanisms of action

Identification of 4E-BP2 forms and their phosphorylation status in ischemia and IR stress.

<p>(A) Samples of the cerebral cortex, C, or hippocampal CA1 region, CA1, from control (SHC and SHC3d) and ischemic animals, without (I15) or with reperfusion (R30 and R3d), were subjected to western blotting with anti-4E-BP2 (4E-BP2), anti-phospho-4E-BP2 Thr³⁷/Thr⁴⁶ (p-Thr37/46) and anti-phospho-rpS6 Ser^235/236 (p-rpS6) antibodies. Arrows show the <i>a</i> and <i>b</i> forms of 4E-BP2 and phospho-rpS6. Figures show representative results. The right numbers represent the apparent MW in kDa from protein markers. (B) Levels of the <i>a</i> and <i>b</i> forms of 4E-BP2 under ischemia and IR. Data are the quantification of the <i>a</i> and <i>b</i> forms (upper and lower bar graphs, respectively) with respect to total 4E-BP2 levels (ratios) and represented in arbitrary units; error bars indicate SEM. p > 0.05, compared with the SHC and SHC3d controls. (C) Quantification of the 4E-BP2 phosphorylation at Thr³⁷/Thr⁴⁶ residues induced by ischemia and reperfusion stress. Data are the quantification of the <i>a</i> and <i>b</i> phospho-forms (upper and lower bar graphs, respectively) detected with anti-phospho-4E-BP2 antibody with respect to total 4E-BP2 levels (ratios) and represented in arbitrary units; error bars indicate SEM. *p < 0.05; **p < 0.01, compared with the controls. All data were from four to six different animals and run in duplicate, and analyzed by ANOVA and ad hoc post-test, unless otherwise stated.</p

4E-BP2 and eIF4G association with eIF4E.

<p>(A) Samples as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121958#pone.0121958.g001" target="_blank">Fig. 1A</a>, were bound to m⁷GTP-Sepharose and analyzed by western blotting with anti-eIF4E (eIF4E), anti-4E-BP2 (4E-BP2), anti-phospho-4E-BP2 Thr³⁷/Thr⁴⁶ (p-Thr37/46) and anti-eIF4G (eIF4G) antibodies. Arrows show the relative position of eIF4E, the <i>a</i> and <i>b</i> forms of 4E-BP2, and eIF4G. The right numbers represent the apparent MW in kDa from protein markers. (B) Quantification of 4E-BP2 and eIF4G bound to eIF4E induced by ischemia and IR stress. No significant differences in the eIF4E levels were found (p ≥ 0.360). Data are the quantification of bound 4E-BP2 (<i>a</i> + <i>b</i> forms; upper bar graph), or bound eIF4G (lower bar graph), with respect to eIF4E levels (ratios) detected with the corresponding antibody and represented in arbitrary units. Error bars indicate SEM. *p < 0.05, compared with the controls; ⁺ p < 0.05, ⁺⁺⁺p < 0.001, cerebral cortex, C, compared with the hippocampal CA1 region, CA1. Differences in eIF4G between R3d and SHC3d groups were done by two-tailed t test of data from six to eight different animals run in duplicate.</p

Analysis of 4E-BP2 immunoprecipitates.

<p>(A) Samples of cerebral cortex, C, or CA1 from control (SHC3d) and ischemic animals with long-term reperfusion (R3d), were immunoprecipitated with anti-4E-BP2 antibody and analyzed by western blotting with: anti-eIF4E antibody and anti-mouse IRDye 680LT-conjugated as secondary antibody (in red); and anti-4E-BP2 antibody and anti-rabbit IRDye 800CW-conjugated (in green). Bar graph shows the quantification of eIF4E in the immunoprecipitates; no significant differences in the 4E-BP2 levels were found (p ≥ 0.520). (B) 4E-BP2 immunoprecipitates of cerebral cortex from R3d and SHC3d control, were subjected to 2-DGE and western blotting (wb) with anti-4E-BP2 antibody (in grey); or were labeled with Cy3- and Cy5-dye, subjected to 2-DGE and after analyzed with an fluorescence imager (Cy images) and by western blotting with anti-4E-BP2 antibody as in (A) (wb, in green). The right numbers represent the apparent MW in kDa from protein markers.</p

Analysis of 4E-BP2 associated to eIF4E by (2-DGE).

<p>Samples from m⁷GTP-Sepharose as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121958#pone.0121958.g003" target="_blank">Fig. 3</a> were subjected to 2-DGE and western blotting and 4E-BP2 spots bound to eIF4E detected with an anti-4E-BP2 antibody (4E-BP2). Figures show the representative results from three different animals. Spots detected in whole samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121958#pone.0121958.g002" target="_blank">Fig. 2A</a>) are shown as control (4E-BP2 control). MW is indicated in kDa.</p

Dissociation of eIF4E-Binding Protein 2 (4E-BP2) from eIF4E Independent of Thr³⁷/Thr⁴⁶ Phosphorylation in the Ischemic Stress Response

<div><p>Eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) are translational repressors that bind specifically to eIF4E and are critical in the control of protein translation. 4E-BP2 is the predominant 4E-BP expressed in the brain, but their role is not well known. Here, we characterized four forms of 4E-BP2 detected by two-dimensional gel electrophoresis (2-DGE) in brain. The form with highest electrophoretic mobility was the main form susceptible to phosphorylation at Thr³⁷/Thr⁴⁶ sites, phosphorylation that was detected in acidic spots. Cerebral ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2 at Thr³⁷/Thr⁴⁶, respectively. The induced phosphorylation was in parallel with the release of 4E-BP2 from eIF4E, although two of the phosphorylated 4E-BP2 forms were bound to eIF4E. Upon long-term reperfusion, there was a decrease in the binding of 4E-BP2 to eIF4E in cerebral cortex, demonstrated by cap binding assays and 4E-BP2-immunoprecipitation experiments. The release of 4E-BP2 from eIF4E was without changes in 4E-BP2 phosphorylation or other post-translational modification recognized by 2-DGE. These findings demonstrated specific changes in 4E-BP2/eIF4E association dependent and independent of 4E-BP2 phosphorylation. The last result supports the notion that phosphorylation may not be the uniquely regulation for the binding of 4E-BP2 to eIF4E under ischemic stress.</p></div

Analysis of 4E-BP2 by two-dimensional gel electrophoresis and changes induced by ischemia and IR stress.

<p>(A) Samples of cerebral cortex from control (SHC and SHC3d) and ischemic animals, without (I15) or with reperfusion (R30 and R3d), were subjected to two-dimensional gel electrophoresis (2-DGE) and western blotting with an anti-4E-BP2 antibody. The antibody-reactive spots were <i>a</i>’, <i>a</i>”, <i>a</i>”’, <i>b</i>’, <i>b</i>”, <i>b</i>”’ and <i>b</i>””. Figures show representative results from six different animals. (B) Identification of the phosphorylation sites for 4E-BP2 spots. R30 sample as in A was subjected to 2-DGE and western blotting for phospho-specific antibodies against 4E-BP2 Thr³⁷/Thr⁴⁶ (p-Thr37/46). The figure shows a representative result from three different animals. Arrows indicate the relative position of the <i>a</i> and <i>b</i> forms of 4E-BP2 in the molecular weight (MW) axis. MW is indicated in kDa.</p

Changes in 4E-BP2/eIF4E association dependent or independent of 4E-BP2 phosphorylation.

<p>Samples of cerebral cortex from control (SHC3d) and ischemic animals with short- and long-term reperfusion (R30 and R3d, respectively), were immunoprecipitated with anti-4E-BP2 antibody and analyzed by western blotting with anti-eIF4E (eIF4E), anti-4E-BP2 (4E-BP2), and anti-phospho-4E-BP1/2 Thr³⁷/Thr⁴⁶ (p-Thr37/46) antibodies. The right numbers represent the apparent MW in kDa from protein markers. The figure shows similar decreased levels of associated eIF4E compared with the control (upper arrows). However, 4E-BP2 was phosphorylated at Thr³⁷/Thr⁴⁶ in R30, and not in R3d (lower arrows).</p