Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant ΔE-torsinA
Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant ΔE-torsinA
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DOI：
10.1021/bi0349569
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Cravatt, BF
中科院分区：
文献类型：
--
作者：
Kustedjo, K;Deechongkit, S;Cravatt, BF
早发性扭转肌张力障碍是一种常染色体显性运动障碍，与蛋白质torsinA（E-302/303; DeltaE-torsinA）中一对谷氨酸残基之一缺失有关。在转染的细胞中，DeltaE-torsinA表现出与野生型（WT）-torsinA相似的生化特性，但表现出不同的亚细胞定位。torsinA的一级结构分析表明，这种蛋白质是ATP结合蛋白AAA家族的膜相关成员。然而，迄今为止，WT-和Δ E-torsinA都没有获得足够的数量和纯度，以允许详细的生物化学和生物物理表征。在这里，我们报告了一个杆状病毒表达系统，提供毫克量的纯化torsin蛋白。发现重组WT-和DeltaE-扭转蛋白A是膜相关糖蛋白，其需要去污剂用于溶解和纯化。WT-和DeltaE-torsinA的生物物理性质的分析表明，这两种蛋白质在溶液中折叠的单体，表现出等效的变性行为下的热和离液（氯化胍）应力。此外，发现两种形式的torsinA显示具有相似k（cat）和K-m值的ATP酶活性。总的来说，这些数据表明torsinA是一种膜相关ATP酶，并表明该蛋白中的DeltaE（302/303）肌张力障碍相关突变不会导致其催化或结构特性的总体变化。这些发现与DeltaE-torsinA通过获得而不是丧失功能促进肌张力障碍的疾病机制一致。本文所述的扭转蛋白A蛋白的重组表达系统应有助于进一步的生物化学和结构研究以测试该假设。
Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E-302/303; DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K-m values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.