Mechanisms of Kinesin Regulation

驱动蛋白调节机制

• 批准号: 8322600
• 负责人: Sarah E. Rice
• 金额: $ 27.75万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322600
• 关键词: ATP phosphohydrolase; Activator Appliances; Active Biological Transport; Address; Binding; Biochemical; Biological; C-terminal; CENP-E protein; Calcium; Cells; Charcot-Marie-Tooth Disease; Chemicals; Chimeric Proteins; Complex; Coupled; Cues; Data; Dependence; Disease; Drosophila genus; Electron Spin Resonance Spectroscopy; Eukaryotic Cell; Family member; Head; Image; Immunoprecipitation; In Vitro; Individual; Kinesin; Label; Life; Light; Maps; Mass Spectrum Analysis; Microtubules; Mitochondria; Molecular Motors; Motor; Movement; Mutation; N-terminal; NMR Spectroscopy; Neurofibromatoses; Neurofibromatosis 2; Occupations; Parkinson Disease; Process; Proteins; Reagent; Regulation; Schizophrenia; Structure; Tail; Testing; Walking; Work; cell motility; cell type; crosslink; flexibility; in vivo; inhibitor/antagonist; mutant; nervous system disorder; response; therapy development

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.

描述（由申请人提供）：分子运动驱动蛋白-1执行大量的运输任务，控制这些过程的调节机制至关重要。驱动蛋白-1的错误调节或驱动蛋白-1货物的错误定位可能涉及几种疾病，如帕金森病、神经纤维瘤病、精神分裂症和腓骨肌萎缩症。驱动蛋白-1的运动机制是很好的理解，我们现在也知道，驱动蛋白-1的C-末端尾部直接相互作用，并抑制头部时，马达是不需要的货物运输。然而，我们不知道驱动蛋白-1调节剂如何启动或停止货物运输。尾部当然也参与其中，因为它与头部、微管和几种不同的驱动蛋白-1激活剂结合，这些激活剂在不同的运输复合物中发挥作用。与尾部分开，Miro蛋白与驱动蛋白-1的酶头部结构域具有直接的Ca 2+依赖性相互作用，并且Miro是Ca 2+依赖性抑制线粒体运动所必需的。我们假设，尾部是一个内在的无序结构域，具有结构灵活性，有利于多个结合伴侣的相互作用，涉及驱动蛋白-1的自身抑制和/或激活，而米罗有一个独特的机制，直接抑制驱动蛋白-1的头抑制线粒体运动的酶机制。为了解决这一假设，我们将首先获得详细的信息，在体外的结构驱动蛋白1的尾巴和它的相互作用与结合伙伴，通过NMR和EPR光谱。我们将确定Miro是否是驱动蛋白-1酶活性的直接的、Ca 2+可切换的抑制剂，使用EPR评估其对驱动蛋白-1机制的影响，并通过交联绘制其与驱动蛋白-1头部的相互作用。在获得尾部和Miro的结构和机制信息后，我们将通过对果蝇S2细胞中的线粒体进行成像来确定它们是否以及如何通过控制体内驱动蛋白-1来影响线粒体运动。这些目标一起将提供一个令人兴奋的新的桥梁之间的体外生物物理和细胞生物学工作的分子马达运输机制。此外，由于Miro和其他驱动蛋白-1调节剂与几种神经系统疾病有关，我们的工作将提供详细的相关生化信息和试剂，从而加速开发治疗方法。