Severity of vanishing white matter disease does not correlate with deficits in eIF2B activity or the integrity of eIF2B complexes

Abstract

Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoencephalopathy vanishing white matter with a wide clinical spectrum. eIF2B comprises five subunits (α-ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide-exchange factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional effects of selected VWM mutations in EIF2B2-5 by coexpressing mutated and wild-type subunits in human cells. The observed functional effects are very diverse, including defects in eIF2B complex integrity; binding to the regulatory α-subunit; substrate binding; and GEF activity. Activity data for recombinant eIF2B complexes agree closely with those for patient-derived cells with the same mutations. Some mutations do not affect these parameters even though they cause severe disease. These findings are important for three reasons; they demonstrate that measuring eIF2B activity in patients' cells has limited value as a diagnostic test; they imply that severe disease can result from alterations in eIF2B function other than defects in complex integrity, substrate binding or GEF activity, and last, the diversity of functional effects of VWM mutations implies that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B, not restoring eIF2B activity.

Identification of Residues That Underpin Interactions within the Eukaryotic Initiation Factor (eIF2) 2B Complex

“…We mutated specific residues in the His-myctagged subunits by site-directed mutagenesis as described previously (16). PCR fragments of NT and I-patch domains as well as truncated forms of eIF2B␥ and ⑀ were cloned into pEBG-6P to produce GST-myc-tagged versions of these fragments.…”

“…Complexes were washed with lysis buffer containing 10% (v/v) glycerol, 0.15% (v/v) Triton X-100, and 20 mM imidazole (for His 6 pull-downs). The purified complexes were either analyzed by Western blot analysis or used to assay eIF2B activity (as described earlier) (16,26).…”

“…18) and that the VWM phenotype is correlated with eIF2B activity (20,21). However, we have recently demonstrated that this is not the case, and in fact, some of the most severe VWM mutations have barely any effect on eIF2B activity (16). Thus, the means by which mutations in the EIF2B1-5 genes cause VWM is not yet understood.…”

“…This provides one important mechanism for regulating eIF2B activity. In the case of mammalian eIF2B, the ␣ subunit is required for full catalytic activity (2,15,16). It is also essential to confer sensitivity to phosphorylated eIF2 (17).…”

“…It is also essential to confer sensitivity to phosphorylated eIF2 (17). Furthermore, although eIF2B␥⑀ "catalytic" subcomplexes do show activity, this is markedly enhanced by the presence of the other subunits (16). Thus, costoichiometric expression of the subunits is necessary for the optimal activity and proper regulation of eIF2B.…”