School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Dis Model Mech. 2017 Sep 1;10(9):1129-1140. doi: 10.1242/dmm.029926. Epub 2017 Aug 2.
Dystonia is the third most common movement disorder, but its diagnosis and treatment remain challenging. One of the most severe types of dystonia is early-onset torsion dystonia (EOTD). The best studied and validated EOTD-associated mutation, torsinAΔE, is a deletion of a C-terminal glutamate residue in the AAA+ ATPase torsinA. TorsinA appears to be an endoplasmic reticulum (ER)/nuclear envelope chaperone with multiple roles in the secretory pathway and in determining subcellular architecture. Many functions are disabled in the torsinAΔE variant, and torsinAΔE is also less stable than wild-type torsinA and is a substrate for ER-associated degradation. Nevertheless, the molecular factors involved in the biogenesis and degradation of torsinA and torsinAΔE have not been fully explored. To identify conserved cellular factors that can alter torsinAΔE protein levels, we designed a new high-throughput, automated, genome-wide screen utilizing our validated torsinA expression system. By analyzing the yeast non-essential gene deletion collection, we identified 365 deletion strains with altered torsinAΔE steady-state levels. One notable hit was , which encodes a member of the protein disulfide isomerase family (PDIs). PDIs reside in the ER and catalyze the formation of disulfide bonds, mediate protein quality control and aid in nascent protein folding. We validated the role of select human PDIs in torsinA biogenesis in mammalian cells and found that overexpression of PDIs reduced the levels of torsinA and torsinAΔE. Together, our data report the first genome-wide screen to identify cellular factors that alter expression levels of the EOTD-associated protein torsinAΔE. More generally, the identified hits help in dissecting the cellular machinery involved in folding and degrading a torsinA variant, and constitute potential therapeutic factors for EOTD. This screen can also be readily adapted to identify factors impacting the levels of any protein of interest, considerably expanding the applicability of yeast in both basic and applied research.
肌张力障碍是第三大常见运动障碍，但诊断和治疗仍然具有挑战性。最严重的肌张力障碍类型之一是早发性扭转痉挛（EOTD）。研究最多和验证最有效的 EOTD 相关突变是 AAA+ATP 酶 torsinA 中 C 末端谷氨酸残基缺失的 torsinAΔE。TorsinA 似乎是内质网（ER）/核膜伴侣，在分泌途径和确定亚细胞结构中具有多种功能。torsinAΔE 变体的许多功能丧失，并且 torsinAΔE 也不如野生型 torsinA 稳定，并且是 ER 相关降解的底物。然而，torsinA 和 torsinAΔE 的生物发生和降解所涉及的分子因素尚未得到充分探索。为了鉴定可以改变 torsinAΔE 蛋白水平的保守细胞因子，我们设计了一种新的高通量、自动化、全基因组筛选，利用我们验证的 torsinA 表达系统。通过分析酵母非必需基因缺失文库，我们鉴定出 365 种改变 torsinAΔE 稳定状态水平的缺失菌株。一个值得注意的命中是 ，它编码蛋白二硫键异构酶家族（PDIs）的成员。PDIs 位于 ER 中，催化二硫键的形成，介导蛋白质质量控制并有助于新生蛋白质折叠。我们在哺乳动物细胞中验证了选择人类 PDIs 在 torsinA 生物发生中的作用，并发现 PDIs 的过表达降低了 torsinA 和 torsinAΔE 的水平。总之，我们的数据报告了首次全基因组筛选，以鉴定改变 EOTD 相关蛋白 torsinAΔE 表达水平的细胞因子。更一般地说，鉴定出的命中有助于剖析涉及 torsinA 变体折叠和降解的细胞机制，并构成 EOTD 的潜在治疗因子。该筛选还可以很容易地适应于鉴定影响任何感兴趣蛋白水平的因素，极大地扩展了酵母在基础和应用研究中的适用性。
