Expression and Characterization of Torsin A

肌张力障碍是一种神经疾病，表现为无法控制的持续性肌肉收缩。早发性肌张力障碍发生在儿童和青少年。早在1997年就发现了导致早发性肌张力障碍的遗传基因。该基因包含了TorsinA的基因。已有研究表明，所有早发性肌张力障碍患者都有该基因的单一损伤，即在编码区3‘端附近缺失一个谷氨酸残基。目前，TorsinA在正常和患病个体中的功能尚不清楚。有关TorsinA的结构信息对于确定其在正常神经功能中的作用将是无价的。此外，确定Glu缺失的后果将有助于深入了解突变体蛋白的异常功能，并可能有助于确定早发性肌张力障碍的治疗方法。这项研究计划的长期目标是确定TorsinA的结构与其在肌张力障碍中的作用之间的关系。这一目标分为几个不同的阶段：o获得TorsinA生物体的高水平表达o确定TorsinA的溶液和/或晶体结构o研究由于Glu302缺失导致TorsinA的结构变化o调查TorsinA中其他突变对神经功能的影响在本R21提案的过程中，我们计划获得TorsinA在细菌、酵母或哺乳动物系统中的高水平表达。一旦确定了TorsinA的合适来源，就可以使用标准的生化技术获得纯蛋白质。将评估这种材料是否适合通过核磁共振波谱复制或X射线衍射法确定其结构。如果获得晶体，我们将开始用晶体照相术确定结构。在晶体生长有问题的情况下，我们将优化溶液条件，以便通过核磁共振光谱确定结构。此外，我们还将研究TorsinA的生化特性，以确定其在神经功能中的作用。

Dystonia is a neurological disease that presents as uncontrolled and sustained muscle contractions. Early onset dystonia occurs in childhood and adolescents. The genetic locus responsible for early onset dystonia was identified in 1997. Contained within this locus is the gene for TorsinA. It has been shown that all individuals possessing early onset dystonia have a single lesion in this gene; the loss of a single Glutamic acid residue near the 3' end of the coding region. Currently the function of TorsinA in normal and affected individuals is unknown. Structural information on TorsinA would be invaluable in defining it role in normal neurological function. In addition, determining the consequences of the Glu deletion will provide insight into the aberrant function of the nutant protein an may aid in defining a treatment for early onset dystonia. The long-term goal of this research plan is to determine the relationship between the structure of TorsinA and its role in dystonia. This goal is divided into a number of distinct phases: o Obtain high levels of expression of TorsinA organisms o Determine the solution and/or crystal structure of TorsinA o Investigate structural changes in TorsinA due to deletion of Glu302 o Investigate the effect on neurological function of other mutations in TorsinA During the course of this R21 proposal we plan on obtaining high-level of expression of TorsinA in bacterial, yeast, or mammalian systems. Once a suitable source of TorsinA is identified, pure protein will be obtained using standard biochemical techniques. The suitability of this material for structure determination by either NMR spectros-copy or X- ray diffraction will be assessed. If crystals are obtained we will initiate structure determination by crystal-lography. In the event that crystal growth is problematic we will optimize solution conditions for structure determination by NMR spectroscopy. In addition, we will also pursue biochemical characterization of TorsinA to determine its role in neurological function.