Mutational analysis of the alpha subunit of eIF2B provides insights into the role of eIF2B bodies in translational control and VWM disease

Abstract

Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlate with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localization of eIF2B to eIF2B bodies was integral for function and whether this localization could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity. Mutational analysis of eIF2Bα showed that missense mutations that disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations that impact the catalytic activity of eIF2B were analyzed, eIF2B bodies were absent and instead eIF2B localized to small foci, termed microfoci. Fluorescence recovery after photobleaching analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analyzed, a diverse impact on localization was observed, which did not seem to correlate with eIF2B activity. These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation.

Optimisation of immunocytochemistry methodology for the detection of endogenous eIF2B localised foci

“…CC-BY-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 8, 2022. ; https://doi.org/10.1101/2022.12.06.519266 doi: bioRxiv preprint Previous characterisation of eIF2B bodies was carried out utilising exogenously expressed GFP tagged proteins (Campbell, Hoyle and Ashe, 2005;Campbell and Ashe, 2006;Egbe et al, 2015;Hodgson et al, 2019;Nuske et al, 2020;Norris et al, 2021). We aimed to detect endogenous eIF2B subunits in U373-MG cells to allow evaluation of endogenous mutant proteins within patient cells or animal models.…”

“…to study the localisation of eIF2B bodies (Campbell, Hoyle and Ashe, 2005;Campbell and Ashe, 2006;Egbe et al, 2015;Hodgson et al, 2019;Nuske et al, 2020;Norris et al, 2021). However, the characterisation of endogenous complexes would allow for analysis of eIF2B bodies in patient samples and animal models of disease.…”

“…Small bodies appear to be largely comprised of catalytic subunits, whereas medium and large bodies include the regulatory subunits (Hodgson et al, 2019). eIF2 shuttles through eIF2B bodies, and fluorescence recovery after photobleaching analysis has shown a correlation between eIF2B GEF activity and the measurement of eIF2 shuttling (Campbell, Hoyle and Ashe, 2005; Hodgson et al 2019; Norris et al ., 2021). In response to stress, eIF2B subunit localisation changes occur, changing the composition of eIF2B bodies, suggesting the presence of subcomplexes which could have an important role in cellular regulation (Wortham et al ., 2014).…”

“…To investigate the role of eIF2B bodies in the cell response to stress, immunocytochemistry (ICC) has been employed to detect and analyse eIF2B bodies in glial cells. In previous studies, GFP tagged subunits have been utilised to study the localisation of eIF2B bodies (Campbell, Hoyle and Ashe, 2005; Campbell and Ashe, 2006; Egbe et al ., 2015; Hodgson et al ., 2019; Nuske et al ., 2020; Norris et al ., 2021). However, the characterisation of endogenous complexes would allow for analysis of eIF2B bodies in patient samples and animal models of disease.…”

Sugar phosphate activation of the stress sensor eIF2B

“…Indeed, yeast eIF2B has been shown to form cellspanning fibrils, although the conditions for formation and ultimate function of these structures are debated 37,38 . Recent work suggests that eIF2Bα is essential for the formation of these eIF2B bodies, and that VWM mutations (including N209Y) disrupt this process 39 . We thus speculate that metabolite binding may be one possible mechanism to modulate these conformational shifts.…”