Enzymatic and Motor Properties of Myosin 19

描述（由申请人提供）：本研究项目的长期目标是了解肌球蛋白XIX（Myo 19）的酶特性、运动特性和细胞功能，肌球蛋白XIX是一种参与线粒体运输和定位的分子运动蛋白，线粒体是多种细胞过程的核心细胞器。线粒体动力学已被证明是部分肌动蛋白为基础的，在其他基因的突变参与线粒体动力学导致人类疾病。此外，微阵列分析表明，与正常组织相比，Myo 19在某些癌症中的表达增加。先前的研究表明，Myo 19在培养细胞中的线粒体运动中起作用，因为GFP标记的Myo 19构建体的表达改变了肺癌细胞系和培养的神经元细胞系中的线粒体动力学。总之，Myo 19蛋白序列的分析和GFP-Myo 19在培养细胞中表达的影响的分析导致Myo 19在线粒体动力学中起作用的假设，因为它包含保守的肌球蛋白运动结构域并通过其尾部结构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Myo 19敲低和过表达功能性和非功能性Myo 19构建体，以确定Myo 19在每个过程中可能发挥的作用。Myo 19运动域的表达和纯化将允许与Myo 19的运动特性相关的速率和平衡常数的完整体外分析。此外，纯化的蛋白质将用于使用体外肌动蛋白滑丝运动测定系统直接检查Myo 19的运动特性。Myo 19在体外的酶和运动特性的检查将允许确定Myo 19运动活性产生力的机制，并确定可能调节其体内功能的因素。通过研究Myo 19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健康和疾病中线粒体动力学的理解。

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.