Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells

Abstract

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2’s alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2αS51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4A391D and the related severe VWM EIF2B4R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.

EIF2B2 mutations in vanishing white matter disease hypersuppress translation and delay recovery during the integrated stress response

“…1). Similar results have been seen with other VWMD alleles, and in different cellular contexts (Kantor et al 2005;van Kollenburg et al 2006b;Sekine et al 2016;Wong et al 2018). The normal levels of bulk translation in VWMD cells occur despite clear data that many VWMD mutations reduce the exchange rate of GDP for GTP on eIF2α (van Kollenburg et al 2006b;Horzinski et al 2010;Liu et al 2011;Wortham and Proud 2015;Wong et al 2018).…”

“…1A). This is similar to previous observations that VWMD cell lines show normal levels of translation in the absence of stress (Kantor et al 2005;van Kollenburg et al 2006b;Sekine et al 2016;Wong et al 2018). Second, western blot analysis showed the examined EIF2B2 mutations did not reduce the levels of EIF2B2 protein (Fig.…”

“…First, fibroblasts derived from VWMD patients with EIF2B4 and EIF2B5 mutations on average displayed an almost significant (P = 0.0500005, Student's ttest) ∼50% reduction in translation activity compared to ∼40% suppression of translation in control cells (Kantor et al 2005). Second, Sekine et al (2016) assessed translation activity in Eif2b4-mutant CHO cells during an acute thapsigargin stress, and these cells also on average exhibited lower translation activity (∼60%-70% suppression in the mutant cell line versus ∼50% suppression in the wild-type cells). Finally, while DTT stress reduced translation to ∼30% in control 293T cell lines, translation was hyperrepressed to ∼15% in isogenic 293T cell lines carrying VWMD mutations in EIF2B1, EIF2B4, or EIF2B5 (Wong et al 2018).…”

An oligomeric state‐dependent switch in the ER enzyme FICD regulates AMP ylation and de AMP ylation of BiP

“…CHO‐K1 cells (ATCC CCL‐61) were phenotypically validated as proline auxotrophs, and their Cricetulus griseus origin was confirmed by genomic sequencing. CHOP :: GFP and XBP1s :: Turquoise reporters were introduced sequentially under G418 and puromycin selection to generate the previously described derivative CHO‐K1 S21 clone (Sekine et al , ). The cells were cultured in Nutrient mixture F‐12 Ham (Sigma) supplemented with 10% ( v / v ) serum (FetalClone II; HyClone), 1 × penicillin–streptomycin (Sigma) and 2 mM l ‐glutamine (Sigma).…”

Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders

“…However it remains unclear how the reduced activity of eIF2B activates the IRE1 and ATF6 branches of the UPR as observed in VWMD patients (van der Voorn et al, 2005; van Kollenburg et al, 2006). Intriguingly, a recent study shows that cells expressing VWMD eIF2B mutants, despite having an enhanced ISR, do not tolerate an additional mutation preventing eIF2α phosphorylation (Sekine et al, 2016), although the mechanisms for this dependence are still unclear. This underlies that the link between VWMD and the UPR is extremely complex and needs further investigations.…”