Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

<p>Abstract</p> <p>Background</p> <p>Eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.</p> <p>Methods</p> <p>We used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of <it>ATF4</it>, and transcriptional induction of stress-specific mRNAs (<it>ATF4, CHOP, ASNS, GRP78</it>) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).</p> <p>Results</p> <p>Despite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.</p> <p>Conclusions</p> <p>Taken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.</p

Effect of eIF2B5 down-regulation.

<p>A. Identical amounts of total cell protein extracted from DDR1 and sh2B5 cells (stably expressing shRNA against eIF2B5 3′UTR) were subjected to Western blot analysis using antibodies specific for eIF2B5 and p38. B. 5×10⁵ cells were labeled with [³⁵S]-Met/Cys mix for 20 minutes followed by protein extraction, TCA-precipitation and scintillation counting of equal amounts of protein. The data represent average of three independent experiments performed in triplicates+/−SE. C. Control (open bars) or sh2B5 cells (dark bars) were incubated with 10 µg/ml Tunicamycin (Tun) for the indicated times, followed by XTT viability assay. Cell viability is expressed as percentages of viable cells grown in Tun-free medium. The data represent average of three independent experiments performed in triplicates+/−SE.</p

Proteome profiling of ER proteins following stress.

<p>The SILAC methodology followed by mass spectrometry of microsomal preparations was processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003783#s4" target="_blank">Materials and Methods</a>. Labeled DDR1 cells and unlabeled sh2B5 or sh2B5+2B5(R195H) were treated with 1 µM Tg for 0, 12 and 24 h. The unlabeled cells at each time point were mixed at a 1∶1 ratio with the labeled DDR1 controls. The level of Bip, PDIA1, PDIA3, PDIA4 and PDIA6 at each time point (except for PDIA6 at baseline in sh2B5+2B5(R195H) cells) relative to DDR1 control cells is shown.</p