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19 （Myo19）的酶学特性、运动特性和细胞功能，肌球蛋白19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部分以肌动蛋白为基础，而参与线粒体动力学的其他基因的突变导致人类疾病。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起作用，因为gfp标记的Myo19构建物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蛋白序列的分析和对培养细胞中GFP-Myo19表达影响的分析得出Myo19在线粒体动力学中起作用的假设，因为它包含一个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Myo19敲除和功能性和非功能性Myo19构建体的过表达来确定Myo19在这些过程中可能发挥的作用。Myo19运动结构域的表达和纯化将允许对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，纯化蛋白将用于使用体外肌动蛋白滑动丝运动测定系统直接检测Myo19的运动特性。在体外检查Myo19的酶和运动特性将允许确定Myo19运动活动产生力的机制，并确定可能调节其体内功能的因素。通过检查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. PUBLIC HEALTH RELEVANCE: Mitochondria play critical roles in healthy cellular function, most importantly the production of energy inside the cell. These "powerhouses" must be able to move to locations within the cell where energy needs are high, and alterations in these transport mechanisms have been associated with human disease. Myosin XIX is a motor protein involved in mitochondrial transport, and studying its motor properties will aid in the understanding of mitochondrial transport in both healthy and disease conditions. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" section above summarizes the final opinions of the committee.