Biochemical effects of mutations in the gene encoding the alpha subunit of eukaryotic initiation factor (eIF) 2B associated with Vanishing White Matter disease.
Biochemical effects of mutations in the gene encoding the alpha subunit of eukaryotic initiation factor (eIF) 2B associated with Vanishing White Matter disease.
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DOI：
10.1186/s12881-015-0204-z
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发表时间：
2015-08-19
影响因子：
--
通讯作者：
Proud CG
中科院分区：
文献类型：
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作者：
Wortham NC;Proud CG
白质消失性白质脑病（Leukoencephalopathy with Vanishing白色Matter，VWM）是一种常染色体隐性遗传疾病，由编码真核生物翻译起始因子eIF 2B的5个亚基的基因EIF 2B 1 -5的种系突变引起。迄今为止，已经对EIF 2B 2 -5基因突变的生化效应进行了分析，但尚未对EIF 2B 1基因突变进行研究。该基因编码eIF 2B α，eIF 2B中最小的亚基，其在eIF 2B复合物的结构和调节中具有重要作用。eIF 2B亚基在HEK 293细胞中过表达，并通过亲和层析从所得细胞裂解物中分离。通过蛋白质印迹评估eIF 2B复合物的形成及其底物eIF 2的结合。还进行了鸟嘌呤核苷酸交换（GEF）活性的测定。在研究的5个eIF 2B α突变中，我们发现3个显示eIF 2B α与复合物其余部分的结合丧失或减少，一个具有增加的GEF活性，另一个没有观察到对活性或复合物形成的影响。这是首次对eIF 2B α VWM突变进行研究。我们发现，一些突变导致预期的GEF活动或复杂的形成，类似于大多数观察到的VWM突变的减少。然而，我们也观察到一些意想不到的变化，这些变化暗示了这些突变对eIF 2B尚未描述的功能的其他影响。
Leukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2Bα, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex. eIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out. Of the 5 eIF2Bα mutations studied, we found 3 that showed loss or reduction of binding of eIF2Bα to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed. This is the first study on eIF2Bα VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.