Candidate NEMO interactors identified by protein microarray screening with full-length NEMO protein.

<p><i>aThe Z-score indicates the how far the average spot intensity for a particular putative interactor fell from the mean of the relevant protein microarray sector spot intensities, measured in standard deviations. A Z-score of greater than four standard deviations (P = 0.002) was deemed significant.</i></p><p><i>bThese proteins are known to interact directly with NEMO, based on the results of tandem affinity purification experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008799#pone.0008799-Bouwmeester1" target="_blank">[17]</a>.</i></p

NEMO interactors influence the transcriptional activation activity NF-kappaB.

<p>(A) Each of the five NEMO interactors was overexpressed in untreated HEK-293T cells and their effect on NF-kappaB transcriptional activation measured by a reporter assay. CALB1, CDK2 and SAG significantly increased reporter activity, indicated by asterisks (n = 4; two-tailed T test; P≤0.05), while other genes had no effect compared to the control vector transfection. Reporter activity is given in relative light units (RLU). (B) CALB1 and CDK2 overexpressed increases NF-kappaB activity in TNFalpha treated cells, while SYT1 overexpression significantly represses activity (n = 4; two-tailed T test; P≤0.05). (C) Confirmation of protein expression following transfection of HEK-293T cells by immunoblot detection of native or epitope-tagged NEMO interactors. Little or no protein expression was detected in the control vector transfected cells. (D) Knockdown of CDK2, SAG and SENP2 in HEK-293T cells mediated by siRNA transfection. RNA levels at the time of NF-kappaB assays were measured by RT-qPCR and are displayed as percentage mRNA remaining after knockdown compared to the amounts present in control siRNA-treated cells. Significant knockdown was seen for both of the genes, as indicated by the asterisks (n = 3; two-tailed T test; P≤0.05). (E) mRNA knockdown of CDK2, SAG and SENP2 reduces NF-kappaB transcriptional activation in TNFalpha stimulated HEK-293T cells, but does not impact upon basal NF-kappaB activity in untreated cells (n = 4; two-tailed T test; P≤0.05).</p

Putative interactors bind to NEMO in GST pulldown, coimmunoprecipitation and mammalian two-hybrid assays.

<p>(A) Immunoblot analysis of GST and GST-NEMO proteins used as control and bait for the pulldown assay. Proteins were detected using anti-GST/HRP conjugate following SDS-PAGE and membrane transfer. (B) Results of GST pulldown assays showing binding of NEMO to putative interactors identified by protein array screening. Each of the interactors and IKKbeta, a known NEMO binder, were overexpressed in transiently transfected HEK-293T cells and the resulting lysates applied to immobilized GST or GST-NEMO. Following incubation and washing, the samples were resolved by SDS-PAGE and the proteins detected using appropriate antibodies. Input lanes were loaded with 5–10% of HEK-293T lysates to confirm protein expression. The size of relevant protein markers is shown beside the blot image. (C–H) Coimmunoprecipitation assays between NEMO and putative binders in HEK-293T cells. Plasmids encoding Xpress-tagged NEMO or the empty parent vector and tagged putative binders were used to transfect HEK-293T cells and the resulting cell lysates used for coimmunoprecipitation assays. For each putative binder, immunoblots are shown for detection of the binder using a tag- or protein-specific antibody, and for detection of Xpress-tagged NEMO. For IKKbeta and each of the five putative interactors, substantial coimmunoprecipitation occurred only in the presence immunoprecipitated NEMO. Input lanes contained 5–10% of the precleared input volume used prior to addition of anti-Xpress antibody. Binding and washing steps were performed in the presence of 0.5% NP-40 for all proteins except SAG, where 0.1% NP-40 was used. (I) NEMO interacts with CALB1, CDK2, SAG, SENP2 and SYT1 in a mammalian two-hybrid system. Empty two-hybrid vectors were cotransfected as a negative control. The MyoD/Id and NEMO/IkappaBalpha protein pairs were used as positive controls, while putative interaction partners cotransfected with empty complementing vector were used as negative controls. For each pair tested, a significant increase (n = 6; two-tailed T test; P≤0.05) in luciferase activity was obtained in partner/NEMO experiments compared to partner/vector experiments (indicated by asterisks).</p

The computational model correctly predicted ATP, AMPK activity and glucose dynamics measured in neurons exposed to glutamate.

<p>(A) Model schematic. State variables are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">Table 1</a>, and reaction numbers correspond to those listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s002" target="_blank">S1 Table</a>. (B-E) Model input and simulations (solid black lines) overlaid on the median and inter-quartile regions (dotted black line, grey shaded area) of previously published fluorescence measurements in single cerebellar granule neurons exposed to glutamate for 10 min [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]. The time of stimulus (model input or glutamate exposure) is marked with a light grey bar. Values were normalised to baseline. (B) A transient (10 min) increase in cytosolic calcium was applied as model input (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#sec008" target="_blank">Methods</a> and d[Cac]/dt equation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s002" target="_blank">S1 Table</a>), and fitted to fluorescence measurements of cytosolic calcium (Fluo-4 AM) in CGNs exposed to glutamate. (C) The simulated ATP dynamics closely aligned with experimental measurements of intracellular ATP concentration [ATeam is a fluorescent reporter of intracellular ATP concentration; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref016" target="_blank">16</a>]]. (D) The simulated transient activation of AMPK resembled experimental measurements of AMPK activity [AMPKAR is a fluorescent reporter of AMPK activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref017" target="_blank">17</a>]]. (E) The model also correctly predicted a prolonged elevation of intracellular glucose and its delayed recovery [Glucose-FRET is a fluorescent reporter of intracellular glucose concentration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref018" target="_blank">18</a>]].</p

Parameter constraints determined from steady-state (ss) analysis.

<p>Parameter constraints determined from steady-state (ss) analysis.</p

Multiple model simulations with varied parameter sets represented the experimental cell-to-cell heterogeneity.

<p>(A-E) Cell-to-cell heterogeneity was modelled by varying parameter values and input characteristics (magnitude and duration of the applied calcium influx) and performing multiple simulations. (A) The median and inter-quartile regions of all model inputs (solid black line within dark grey region) well resembled the median and inter-quartile regions (black dotted line within light grey region) of previously published fluorescent measurements of cytosolic calcium [Ca_c; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]]. (B-E) For each simulation, the metrics shown in (B-E) were calculated. The coloured data points link these simulations across the figures. The green data points were predicted with the parameter set as listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">2</a>. The parameter sets predicting the yellow, cyan and red data points are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.s003" target="_blank">S2 Table</a>. (B,C) The predicted variability in the (B) maximum fold change and (C) recovery duration of the model input (black box) closely matched experimental measurements (white box). Recovery duration was calculated as the time taken for the signal to recover to ±2% of baseline signal. (D) Box- and scatter-plots of the minimum ATP, maximum AMPK activity and maximum glucose fold changes during the excitotoxic stimulus, calculated from multiple model predictions and experimental measurements (Exp.) from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]. (E) Box- and scatter-plots of the post-excitotoxicity recovery duration of the ATP, AMPK activity and glucose levels as calculated from model predictions and experimental measurements (Exp.) from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>].</p

Steady-state concentrations of modelled state variables.

<p>Steady-state concentrations of modelled state variables.</p

Proposed Charge Transfer Network in Bak.

<p>(A) Sequence alignment of central helices of Bak and Bax. An asterisk indicates a single, fully conserved residue. A colon indicates conservation between groups of strongly similar biochemical properties. A period indicates conservation between groups of weakly similar biochemical properties. The residues involved in the charge transfer network in Bax are conserved in Bak as Trp125, Arg127 and Arg137. (B) Bak structure (ochre) is displayed according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002565#pcbi-1002565-g005" target="_blank">Figure 5B</a>, with the same top view of the central helix, and the same color coding for C-domain (yellow), BH3-domain (cyan), central helix (green) and hub residues (red and blue). Residues Trp125, Arg127 and Arg137 are organized in a similar manner to their Bax homologues, suggesting that they may also play an essential role during Bak activation.</p

Proposed charge transfer network in Bax, indicated by net changes in residue charges.

<p>The information of the Bim-SAHB induced activation of Bax is transmitted from the Bax activation site via a charge transfer network through the core of the Bax protein, up to the Bax C- and BH3-domains. Inside the hydrophobic core of Bax, the central helix, helix 5, acts as a hub which collects and distributes charge density, mainly through residues Trp107, Arg109 and Lys119. The color coding from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002565#pcbi-1002565-g001" target="_blank">Figure 1</a> is maintained. Additionally, helix 5 is highlighted in green. The Bax residues which transfer an amount of charge of one standard deviation higher than average (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002565#pcbi.1002565.s003" target="_blank">Table S1</a>) are explicitly displayed and color coded according to whether they become more positive (blue) or negative (red) upon activation. (A) The residues which transfer a significant amount of charge were found at the Bax activation site, on the loop 1–2, inside the BH groove holding the Bax C-domain, and at one end of the C-domain (see also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002565#pcbi.1002565.s001" target="_blank">Figure S1</a>). Additionally, several such residues were found on helix 5, the central helix in Bax, and on the Bax BH3 domain, suggesting that the interaction at the Bax activation site is transmitted via a network of charges from the activation site, through the protein core, to the C- and BH3-domains. (B) Top view of helix 5 is given. The organization of residues Trp107, Arg109 and Lys119 inside the hydrophobic core of Bax suggests that helix 5 acts as a charge transfer hub, which integrates and distributes charge density.</p

Sensitivity analysis indicated that glucose import dynamics are critical to the post-excitotoxic glucose recovery.

<p>(A-C) Parameters were varied by 0.5 (navy), 0.75 (light blue), 1 (green), 1.5 (orange) and 2 (red) times the steady-state values listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.t002" target="_blank">2</a>, and the effect was calculated for the post-excitotoxic recovery duration of the (A) ATP, (B) AMPK activity and (C) Glucose signals. The varied parameter is written under each bar chart. The 5 parameters with the greatest effect on each metric are shown (left to right in order of effect), along with other parameters mentioned in the text. Data were omitted for parameter values at which the modelled state variables did not return to baseline within the simulation time (100 min). (D, E) Experimental traces (top panels) and multiple model simulations (bottom panels) of intracellular glucose concentration with either (D) glucose import or (E) AMPK inhibited prior to exposure to a transient excitotoxic stimulus. (D) Glucose import was inhibited by exposure to Cytochalasin B or by reduction of the modelled glucose import kinetics (Rx 9). (E) AMPK activity was inhibited by exposure to Compound C or by reduction of the modelled AMPK phosphorylation kinetics (Rx 6). Compound C experiments have been published previously [Fig 6A from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148326#pone.0148326.ref015" target="_blank">15</a>]]. (F) Box- and scatter-plots of the glucose recovery duration with and without glucose import or AMPK inhibition (* ranksum p < 0.05).</p