Enzymatic and Motor Properties of Myosin 19

描述(申请人提供)：这项研究项目的长期目标是了解肌球蛋白XIX(Myo19)的酶特性、运动特性和细胞功能，Myo19是一种参与线粒体运输和定位的分子马达蛋白，线粒体是多种细胞过程的中心细胞器。线粒体动力学已被证明部分以肌动蛋白为基础，参与线粒体动力学的其他基因的突变会导致人类疾病。此外，微阵列分析表明，与正常组织相比，Myo19在某些癌症组织中的表达增加。先前的研究表明，Myo19在培养细胞的线粒体运动中发挥作用，因为GFP标记的Myo19结构的表达改变了肺癌细胞系和培养的神经元细胞系的线粒体动力学。综上所述，对Myo19蛋白序列的分析以及对GFP-Myo19在培养细胞中表达的影响的分析，导致了Myo19在线粒体动力学中发挥作用的假设，因为它含有保守的肌球蛋白运动域，并通过其尾部域与线粒体相互作用。我们将在细胞分裂和凋亡的细胞培养模型中使用Myo19的下调和过表达功能和非功能的Myo19结构来确定Myo19在每个过程中可能扮演的角色。Myo19运动域的表达和纯化将允许对与Myo19运动特性相关的速率和平衡常数进行完整的体外分析。此外，纯化的蛋白质将用于使用体外肌动蛋白滑动细丝运动测试系统直接检测Myo19的运动特性。在体外检测Myo19的酶和运动特性将有助于确定Myo19运动产生力量的机制，并确定可能调节其在体内功能的因素。通过研究Myo19的功能作用、酶特性和运动特性，这些拟议的研究将进一步加深我们对线粒体动力学在健康和疾病中的理解。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the enzymatic properties, motor properties, and cellular functions of myosin XIX (Myo19), a molecular motor protein involved in the transport and localization of mitochondria, an organelle central to multiple cellular processes. Mitochondrial dynamics have been shown to be partially actin-based, and mutations in other genes involved in mitochondrial dynamics lead to human diseases. Additionally, microarray analyses indicate that Myo19 expression is increased in certain cancers, compared to normal tissues. Previous studies show that Myo19 plays a role in mitochondrial motility in cultured cells as expression of GFP-tagged Myo19 constructs alters mitochondrial dynamics in a pulmonary cancer cell line and in a cultured neuronal cell line. Taken together, analysis of the Myo19 protein sequence and analysis of the effect of GFP-Myo19 expression in cultured cells lead to the hypothesis that Myo19 plays a role in mitochondrial dynamics, as it contains a conserved myosin motor domain and interacts with mitochondria via its tail domain. We will use both Myo19 knock-down and overexpression of functional and non-functional Myo19 constructs in cell culture models of cell division and apoptosis to determine what role Myo19 may play in each of those processes. Expression and purification of Myo19 motor domain will allow for a complete in vitro analysis of the rate and equilibrium constants associated with the motor properties of Myo19. Additionally, purified protein will be used to directly examine the motor properties of Myo19 using an in vitro actin sliding filament motility assay system. Examination of both the enzymatic and motor characteristics of Myo19 in vitro will allow the determination of the mechanism by which Myo19 motor activity generates force, and determination of factors that may regulate its function in vivo. By examining the functional roles, enzymatic properties, and motor properties, of Myo19, these proposed studies will further our understanding of mitochondrial dynamics in health and disease.