Date Published: January 15, 2013
Publisher: Public Library of Science
Author(s): Rogerio A. de Almeida, Anne Fogli, Marina Gaillard, Gert C. Scheper, Odile Boesflug-Tanguy, Graham D. Pavitt, Eric Jan. http://doi.org/10.1371/journal.pone.0053958
Recessive inherited mutations in any of five subunits of the general protein synthesis factor eIF2B are responsible for a white mater neurodegenerative disease with a large clinical spectrum. The classical form is called Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM). eIF2B-related disorders affect glial cells, despite the fact that eIF2B is a ubiquitous protein that functions as a guanine-nucleotide exchange factor (GEF) for its partner protein eIF2 in the translation initiation process in all eukaryotic cells. Decreased eIF2B activity measured by a GEF assay in patients’ immortalised lymphocytic cells provides a biochemical diagnostic assay but is limited by the availability of eIF2 protein, which is classically purified from a mammalian cell source by column chromatography. Here we describe the generation of a recombinant expression system to produce purified human eIF2 from yeast cells. We demonstrate that human eIF2 can function in yeast cells in place of the equivalent yeast factor. We purify human eIF2 and the C-terminal domain of human eIF2Bε using affinity chromatography from engineered yeast cells and find that both function in a GEF assay: the first demonstration that this human eIF2Bε domain has GEF function. We show that CACH/VWM mutations within this domain reduce its activity. Finally we demonstrate that the recombinant eIF2 functions similarly to eIF2 purified from rat liver in GEF assays with CACH/VWM eIF2B-mutated patient derived lymphocytic cells.
Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM) (OMIM #603896) was described in the 1990s , . It is a fatal childhood onset white matter disease with a chronic progressive course exacerbated by acute episodes , . Inherited mutations in any of the five genes encoding subunits of the general protein synthesis initiation factor eIF2B (EIF2B1-5) cause CACH/VWM , . The subsequent description of a large clinical spectrum of the disease from neonatal to adult onset or even asymptomatic forms led to the concept of eIF2B-related disorders that are recognized by peculiar magnetic resonance imaging (MRI) abnormalities . Well over 100 different, mainly missense, mutations have been presently reported . Their consequences on the eIF2B complex have been demonstrated in yeast and in humans , , . Abnormalities in glial cell development have been suggested by studies of patient samples , , , , , ,  and studies of a mouse model . One recent suggested explanation is that altered expression of splicing regulatory factors in eIF2B mutated glial cells may cause altered splicing regulation of the important myelin proteins PLP and DM20 . However many aspects of the disease are still not understood and no current therapy is available.