Mechanism of spastin-mediated microtubule severing

描述（由申请人提供）：Spastin是一种AAA ATP酶，在遗传性痉挛性截瘫（HSP）中发生突变。HSP的特征是脊髓长轴突的神经变性，最终导致轮椅使用和膀胱控制困难。最近的研究表明，痉挛素切断微管。细胞骨架似乎在维持神经健康方面起着重要作用，因为微管马达（如KIF5a和动力蛋白）的突变也会导致神经退行性疾病。与微管末端动力学的丰富信息相反，关于痉挛素使微管内部断裂的机制一无所知。到目前为止，已经确定痉挛蛋白组装成六聚体环，并且突出到环孔中的环识别微管蛋白的C末端氨基酸。关键孔环氨基酸是切断所需的，包括由疾病相关突变改变的氨基酸。Spastin还含有第二个微管结合结构域，其与微管产生独特的相互作用，并且是切断所需的。本研究将探讨这些先前的研究结果所提出的问题，如痉挛是否通过转运微管蛋白通过中央孔，如何孔环促进切断和微管痉挛绑定。将通过使用GroEL陷阱和天然凝胶电泳或凝胶过滤色谱法评估解折叠。将制备具有孔环突变和ATP酶缺陷突变体的混合寡聚体，以检查孔环在切断中的作用。化学交联将用于绘制微管上痉挛蛋白结合的位置。这项工作将是重要的，在了解更多关于神经变性的HSP和轴突的维护一般。遗传性痉挛性截瘫（HSP）是一种遗传性疾病，其中脊髓中最长的神经随着时间的推移而退化，导致无法行走。该项目将使用生物化学来了解导致HSP的基因之一Spastin的工作方式。了解痉挛如何工作可能会导致HSP的治疗，并增加对所有身体如何保持健康神经的理解。

DESCRIPTION (provided by applicant): Spastin, a AAA ATPase, is mutated in Hereditary Spastic Paraplegia (HSP). HSP is characterized by neurodegeneration of the long axons of the spinal cord, leading eventually to wheelchair use and difficulty with bladder control. Recently it has been shown that spastin severs microtubules. The cytoskeleton appears to play an important role in maintaining neural health, as mutations in microtubule motors, such as KIF5a and dynein, also cause neurodegenerative disorders. In contrast to the wealth of information regarding microtubule end dynamics, nothing is known about the mechanism by which spastin makes internal breaks in microtubules. So far, it has been determined that spastin assembles into a hexameric ring and that loops projecting into the pore of the ring recognize the C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. Spastin also contains a second microtubule binding domain which makes a distinct interaction with microtubules and is required for severing. This study will explore questions raised by these previous findings, such as whether spastin unfolds tubulin by translocating it through the central pore, how the pore loops promote severing and where on the microtubule spastin binds. Unfolding will be assessed by use of a GroEL trap and native gel electrophoresis or gel filtration chromatography. Mixed oligomers with pore loop mutations and ATPase deficient mutants will be made to examine the role of the pore loops in severing. Chemical crosslinking will be used to map the location on the microtubule where spastin binds. This work will be important in learning more about neurodegeneration in HSP and axonal maintenance in general. Hereditary Spastic Paraplegia (HSP) is an inherited disease in which the longest nerves in the spinal cord degenerate over time, leading to an inability to walk. This project will use biochemistry to understand the way in which spastin, one of the genes that causes HSP, works. Understanding how spastin works may lead to treatments for HSP and an increased understanding of how all bodies maintain healthy nerves.