Mechanisms of Kinesin Regulation

描述（由申请人提供）：分子马达驱动蛋白-1执行大量运输任务，控制这些过程的调节机制是至关重要的。肌动蛋白-1的错误调控或肌动蛋白-1货物的错误定位可能与几种疾病有关，如帕金森病、神经纤维瘤病、精神分裂症和沙克-玛丽-图斯病。Kinesin-1的运动机制已经被很好地理解，我们现在也知道，当不需要马达进行货物运输时，Kinesin-1的c端尾部直接与头部相互作用并抑制头部。然而，我们不知道驱动蛋白-1调节因子如何启动或停止货物运动。尾巴当然参与其中，因为它与头部、微管和几种不同的运动蛋白-1激活剂结合，这些激活剂在不同的运输复合物中起作用。从尾部分离，Miro蛋白与激酶-1的酶头结构域有直接的Ca2+依赖性相互作用，Miro是Ca2+依赖性线粒体运动抑制所必需的。我们假设尾部是一个内在的无序结构域，具有结构灵活性，促进参与激酶-1自抑制和/或激活的多个结合伙伴相互作用，而Miro具有独特的机制，直接抑制激酶-1头部的酶促机制，抑制线粒体运动。为了解决这一假设，我们将首先在体外通过核磁共振和EPR光谱获得有关运动蛋白-1尾部结构及其与结合伙伴相互作用的详细信息。我们将确定Miro是否是一种直接的、Ca2+可切换的激酶1酶活性抑制剂，使用EPR评估其对激酶1机制的影响，并通过交联绘制其与激酶1头的相互作用。在获得尾巴和Miro的结构和机制信息后，我们将通过对果蝇S2细胞线粒体成像，确定它们是否以及如何通过控制体内的kinesin-1来影响线粒体运动。这些目标将在分子运动运输机制的体外生物物理学和细胞生物学工作之间提供一个令人兴奋的新桥梁。此外，由于Miro和其他驱动蛋白-1调节因子与几种神经系统疾病有关，我们的工作将提供详细的、相关的生化信息和试剂，从而加快开发治疗方法的努力。

DESCRIPTION (provided by applicant): The molecular motor kinesin-1 performs a large number of transport tasks, and the regulatory mechanisms governing those processes are critical. Mis-regulation of kinesin-1 or mis-localization of kinesin-1 cargoes may be implicated in several diseases such as Parkinson's disease, neurofibromatosis, schizophrenia, and Charcot-Marie-Tooth disease. Kinesin-1's motile mechanism is well understood, and we now also know that kinesin-1's C-terminal tail interacts directly with and inhibits the heads when the motor is not needed for cargo transport. However, we do not know how kinesin-1 regulators initiate or stop cargo movement. The tail is certainly involved, as it binds to heads, microtubules, and several distinct kinesin-1 activators that function in different transport complexes. Separate from the tail, the Miro protein has a direct, Ca2+dependent interaction with kinesin-1's enzymatic head domains, and Miro is required for Ca2+dependent suppression of mitochondrial motility. We hypothesize that the tail is an intrinsically disordered domain, having structural flexibility that facilitates multiple binding partner interactions involved in kinesin-1 auto-inhibition and/or activation, while Miro has a distinct mechanism, directly inhibiting the enzymatic mechanism of kinesin-1 heads to suppress mitochondrial movement. To address this hypothesis, we will first gain detailed information in vitro about the structure of the kinesin-1 tail and its interactions with binding partners, by NMR and EPR spectroscopy. We will determine whether Miro is a direct, Ca2+switchable inhibitor of kinesin-1's enzymatic activity, assess its effects on kinesin-1 mechanism using EPR, and map its interaction with kinesin-1 heads by cross-linking. After obtaining this structural and mechanistic information on both the tails and Miro, we will determine whether and how they influence mitochondrial movement by controlling kinesin-1 in vivo, by imaging mitochondria in live Drosophila S2 cells. These Aims together will provide an exciting new bridge between in vitro biophysical and cell biological work on molecular motor transport mechanisms. Furthermore, as Miro and other kinesin-1 regulators have been implicated in several neurological diseases, our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.