Mechanisms of Lis1 and NudE/L Regulation of the Molecular Motor Dynein

项目总结/摘要 基于微管的运输是细胞分裂、细胞迁移和许多细胞因子的运输所必需的。 货物。许多神经发育和神经退行性疾病是由以下疾病引起的或与以下疾病有关： 微管转运受损。细胞质动力蛋白1是两种分子马达蛋白之一 负责微管运输的细胞。动力蛋白是一种高度调节的马达， 调节其功能和活性的接头蛋白的数量。突变或拷贝数变异 动力蛋白调节蛋白也导致神经发育疾病。尽管对人类的重要性 然而，在健康方面，动力蛋白如何调节的机制在很大程度上是未知的。NIH K99/R 00的提案 独立之路奖旨在了解Lis 1和NudE/L，这两个监管机构需要 几乎所有动力蛋白的功能，调节动力蛋白的活性。受损的Lis 1和NudE/L功能涉及一种 许多人类疾病，包括小头畸形、无脑畸形、精神分裂症和自闭症。在目标1中， 在该奖项的指导阶段，DeSantis博士将确定Lis 1和NudE/L如何改变动力蛋白功能， 结构生物学和纯蛋白质重组实验的结合。动力蛋白的磷酸化， Lis 1和NudE/L改变了它们的活性，但其发生机制尚不清楚。在独立期间 DeSantis博士还将确定翻译后修饰如何影响动力蛋白， Lis 1和NudE/L活性。在目标2中，DeSantis博士将使用一种新的动力蛋白调节途径来确定新的动力蛋白调节途径。 蛋白质组学、细胞生物学、活细胞成像和重组蛋白质重构的组合。德桑蒂斯博士 已经鉴定了新的Lis 1和NudE/L相互作用蛋白，并将阐明它们的功能， 在指导和独立授标阶段，这项工作的结果将揭示 动力蛋白的调节机制，这对人类健康和疾病有深远的影响。博士 DeSantis将在该奖项的K99部分接受冷冻电子显微镜培训。当结合 她在生物化学、细胞生物学和活细胞成像方面的背景，学习冷冻电子显微镜将 使DeSantis博士关于运动蛋白调节机制的研究远远超出了 K99/R 00奖。

PROJECT SUMMARY/ABSTRACT Microtubule-based transport is required for cell division, cell migration, and for transport of a number of cellular cargoes. A number of neurodevelopmental and neurodegenerative diseases are caused by or associated with impaired microtubule-based transport. Cytoplasmic dynein 1 (dynein) is one of two molecular motor proteins that are responsible for microtubule-based transport. Dynein is a highly regulated motor and interacts with a number of adaptor proteins that modulate its function and activity. Mutations or copy number variations of dynein regulatory proteins also leads to neurodevelopmental diseases. Despite the importance to human health, mechanisms of how dynein is regulated are largely unknown. This proposal for an NIH K99/R00 Pathway to Independence Award seeks to understand how Lis1 and NudE/L, which are two regulators required for nearly every dynein function, modulate dynein activity. Impaired Lis1 and NudE/L function is implicated in a number of human diseases, including microcephaly, lissencephaly, schizophrenia, and autism. In Aim1 during the mentored phase of the award, Dr. DeSantis will determine how Lis1 and NudE/L alter dynein function using a combination of structural biology and pure protein reconstitution experiments. Phosphorylation of dynein, Lis1, and NudE/L alter their activity but the mechanism of how this occurs in unknown. During the independent phase of the award, Dr. DeSantis will also determine how post-translational modifications influence dynein, Lis1, and NudE/L activity. In Aim 2, Dr. DeSantis will identify novel dynein regulatory pathways using a combination of proteomics, cell biology, live cell imaging, and recombinant protein reconstitutions. Dr. DeSantis has already identified novel Lis1 and NudE/L interacting proteins and will elucidate their function and mechanism during the mentored and independent award phase. The results of this work will reveal mechanisms of dynein regulation, which has far reaching implications in human health and disease. Dr. DeSantis will receive training in cryo-electron microscopy in the K99 portion of the award. When combined with her background in biochemistry, cell biology, and live cell imaging, learning cryo-electron microscopy will empower Dr. DeSantis' research about mechanisms of motor protein regulation far beyond the duration of the K99/R00 award.