The small molecule ISRIB rescues the stability and activity of Vanishing White Matter Disease eIF2B mutant complexes

  1. Yao Liang Wong  Is a corresponding author
  2. Lauren LeBon
  3. Rohinton Edalji
  4. Hock Ben Lim
  5. Chaohong Sun
  6. Carmela Sidrauski  Is a corresponding author
  1. Calico Life Sciences LLC, United States
  2. AbbVie, United States
4 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
eIF2B isoforms have identical GEF activity and ISRIB alters the sensitivity of eIF2B to eIF2 phosphorylation.

(A) Coomassie-stained SDS-PAGE gel of recombinant human eIF2 used as a substrate in the in vitro GEF assays. (B) Multi-angle light scattering analysis of purified eIF2, indicating good agreement …

https://doi.org/10.7554/eLife.32733.002
Figure 1—figure supplement 1
Purification of eIF2B mutants.

(A) Primary sequence of the eIF2B δ1 and δ2 isoforms, showing the region in which they differ at the N-terminus of the protein. (B) Coomassie-stained SDS-PAGE gels of purified recombinant human …

https://doi.org/10.7554/eLife.32733.003
Figure 2 with 2 supplements
Recombinant eIF2B VWMD mutants have reduced GEF activity and complex stability that is rescued by ISRIB.

(A) VWMD mutations tested in this study, visualized as spheres on the structure of S. pombe eIF2B (PDB: 5B04; Kashiwagi et al., 2016). The human mutation sites are shown in plain text and the …

https://doi.org/10.7554/eLife.32733.004
Figure 2—figure supplement 1
Recombinant eIF2B VWMD mutants have reduced GEF activity that is rescued by ISRIB.

(A) All eIF2B VWMD mutant complexes except εR136H have reduced GEF activity compared to WT. ISRIB dose-response curves of GDP release half-life for each mutant. Half-lives of GDP release at each …

https://doi.org/10.7554/eLife.32733.005
Figure 2—figure supplement 2
Recombinant eIF2B VWMD mutants have reduced complex stability that is rescued by ISRIB.

(A–B) Immunoblotting of eIF2Bδ (representative of the 4mer subcomplex) and eIF2Bα from the SEC experiments in (A) Figure 2E and (B) Figure 3B. Bands shown are simulated representations of the …

https://doi.org/10.7554/eLife.32733.006
ISRIB-resistant mutants in the δ subunit can exhibit WT or VWMD-like stability and GEF activity.

(A) ISRIB-resistant mutations tested in this study, visualized as spheres on the structure of S. pombe eIF2B (PDB: 5B04; Kashiwagi et al., 2016). The human mutation sites are shown in plain text and …

https://doi.org/10.7554/eLife.32733.008
Figure 4 with 4 supplements
ISRIB attenuates the ISR and rescues the GEF activity of eIF2B in HEK293T VWMD mutant cells.

(A) Levels of the five eIF2B subunits were measured by immunoblotting (Wes analysis) of lysates from WT and VWMD cell lines. The chemiluminescence signal of each subunit was normalized to α-tubulin …

https://doi.org/10.7554/eLife.32733.009
Figure 4—figure supplement 1
Several VWMD mutations reduce the protein level of the affected subunit in cells.

(A) Schematic of the workflow used to generate VWMD mutant HEK293T cell lines. Cells were co-transfected with a Cas9-OFP-gRNA plasmid and a ssDNA HDR template targeting a specific mutation. …

https://doi.org/10.7554/eLife.32733.010
Figure 4—figure supplement 2
VWMD mutant cells have normal rates of protein synthesis and ISRIB partially rescues a stress-induced reduction.

(A) ISRIB blocks ATF4 induction in VWMD mutants. Immunoblot (Wes analysis) of endogenous ATF4 protein levels in WT and VWMD mutant cells. Total eIF2α was used as a loading control. Cells were …

https://doi.org/10.7554/eLife.32733.011
Figure 4—figure supplement 3
VWMD mutant cells show a similar response to ER stress as WT cells.

Tg dose-response curves of ATF4-luciferase reporter activity in WT and VWMD cell lines. Cells were treated with increasing concentrations of Tg for 7 hr (N = 3, mean ± SD). The values for δR483W …

https://doi.org/10.7554/eLife.32733.012
Figure 4—figure supplement 4
Lysates from VWMD cells exhibit reduced GEF activity in vitro that is enhanced by ISRIB.

(A) Levels of the five eIF2B subunits were measured by immunoblotting (Wes analysis) of lysates from different cell lines. The cell lines were: HEK293T (human embryonic kidney, SV40-transformed), …

https://doi.org/10.7554/eLife.32733.013

Tables

Table 1
eIF2B mutants generated in this study
https://doi.org/10.7554/eLife.32733.007
eIF2B subunitMutationAge of disease onset (years)Disease allelesHEK293T cells generatedRecombinant protein generated
αV183F10–17Homozygous*XX
γI346T1–4Homozygous; also compound heterozygous with G47EX-
δR483W<1HomozygousXX
εR113H1–30Homozygous; also compound heterozygous with multiple other mutations§XX
εR136H3Homozygous#-X
εR195H<1Homozygous**XX
δL179FISRIB-resistant; not naturally occurring††-X
δL487WISRIB-resistant; not naturally occurring-X
  1. *Ohlenbusch et al., 2005

    Wu et al., 2009

  2. van der Knaap et al., 2003

    §Fogli et al., 2004

  3. #Kantor et al., 2005

    **Fogli et al., 2002

  4. ††Sekine et al., 2015

Key resources table
Reagent type or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (H. sapiens)HEK293T with ATF4-Luc reporterPMID: 23741617
Cell line (H. sapiens)HEK293T (eIF2Bα V183F) with ATF4-Luc reporterThis papertwo clones generated
Cell line (H. sapiens)HEK293T (eIF2Bγ I346T) with ATF4-Luc reporterThis papertwo clones generated
Cell line (H. sapiens)HEK293T (eIF2Bδ R483W) with ATF4-Luc reporterThis papertwo clones generated
Cell line
(H. sapiens)
HEK293T (eIF2Bε R113H) with ATF4-Luc reporterThis papertwo clones generated
Cell line (H. sapiens)HEK293T (eIF2Bε R195H) with ATF4-Luc reporterThis papertwo clones generated
AntibodyRabbit monoclonal anti-ATF4Cell Signaling#11815(1:50) in Wes
AntibodyRabbit monoclonal anti-eIF2αCell Signaling#5324(1:100) in Wes
AntibodyRabbit polyclonal anti-eIF2BαProteinTech#18010–1-AP(1:50) in Wes
AntibodyRabbit polyclonal anti-eIF2BβProteinTech#11034–1-AP(1:50) in Wes
AntibodyRabbit polyclonal anti-eIF2BγProteinTech#11296–2-AP(1:25) in Wes
AntibodyRabbit polyclonal anti-eIF2BδProteinTech#11332–1-AP(1:50) in Wes
AntibodyRabbit polyclonal anti-eIF2BεBethyl Labs#A302-556(1:50) in Wes
AntibodyMouse monoclonal anti-tubulinCell Signaling#3873(1:50) in Wes
Recombinant DNA reagentCRISPR nuclease vector with OFP reporterThermo Fisher#A21174
Sequence-based reagenteIF2Bα V183F guide RNAThis paperGTGGTGCTAGATGCTGCTGTCGG
Sequence-based reagenteIF2Bγ I346T guide RNAThis paperTGACAATCTGGGCTGACGAATGG
Sequence-based reagenteIF2Bδ R483W guide RNAThis paperGACTAGATTCAACAACCGTAGGG
Sequence-based reagenteIF2Bε R113H guide RNAThis paperCCGCCCTACATCTCTCAATGTGG
Sequence-based reagenteIF2Bε R195H guide RNAThis paperTTGTCTTCGTGGCAACGAGTTGG
Recombinant proteinGST-PERKThermo Fisher#PV5106Used to phosphorylate eIF2in vitro
Commercial assayONE-GLO luciferase assayPromega#E6120
Chemical compoundBodipy-FL-GDPThermo Fisher#G22360
Chemical compoundISRIBPMID: 23741617Synthesized in-house
Chemical compoundThapsigarginSigma-Aldrich#T9033Stock solution prepared in DMSO
Chemical compoundTunicamycinSigma-Aldrich#T7765Stock solution prepared in DMSO

Additional files

Supplementary file 1

Allele sequences of HEK293T mutant cell lines used in this study.

https://doi.org/10.7554/eLife.32733.014
Transparent reporting form
https://doi.org/10.7554/eLife.32733.015

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