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Enzymatic and Motor Properties of Myosin III

肌球蛋白 III 的酶学和运动特性

基本信息

批准号：
7384963
负责人：
CHRISTOPHER M YENGO
金额：
$ 7.92万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2009-05-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7384963
关键词：
Actins Active Sites Affect Affinity Award Binding Binding Sites Biochemical Biological Biological Assay Biology C-terminal COS Cells California Cells Cellular biology Characteristics Collaborations Complement Crystallography Cytoskeleton Data Defect Development Disruption Down-Regulation Drosophila melanogaster Electron Microscopy Equipment F-Actin Family Family member Funding Genes Goals Hair Cells Hearing Hela Cells Human In Vitro Invertebrates Kinetics Laboratories Labyrinth Lead Length Link Mediating Medical center Microfilaments Molecular Motors Motor Motor Activity Motor Neurons Movement Mutate Mutation Myosin ATPase Myosin III N-terminal North Carolina Organ PAK-1 kinase Pennsylvania Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Photoreceptors Phototransduction Physiological Play Point Mutation Process Property Protein Dephosphorylation Proteins Publications Rate Regulation Research Research Personnel Research Project Grants Research Proposals Resolution Retinal Retinal Degeneration Role Scientist Sensory Sensory Process Site Stereocilium Structure Tail Technology Testing United States National Institutes of Health Universities Vertebrates Virginia Vision Work Xenopus base cell growth regulation cell motility crosslink deafness hearing impairment in vivo interest member novel prevent research study single molecule

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the enzymatic and motor properties of myosin III that allow it to play a critical role in sensory cells. Disruption of the myosin IIIA gene results in deafness in vertebrates as well as retinal degeneneration and phototransduction defects in invertebrates. Myosin III is an actin-based molecular motor that contains a conserved motor domain characteristic of the myosin superfamily, but also contains an N-terminal kinase domain. We propose that kinase activity is activated by phosphorylation of the kinase domain activation loop which stimulates autophosphorylation of loop 2 in the motor domain and results in downregulation of motor activity. Phosphatase activity returns the motor to its active state and inactivates the kinase domain. We will examine the enzymatic, motile, and structural properties of myosin IIIA constructs with the kinase domain activated/inactivated, as well motor domain activated/inactivated. Myosin IIIA is different from myosin IIIB in that it contains an actin-binding motif in its C-terminal tail. We propose that the additional actin binding site in the tail allows myosin IIIA to cross-link actin filaments, cooperatively activate the motor enzymatic cycle, and enhances its motile properties by increasing the overall affinity of myosin IIIA for actin. We will directly determine the role of the tail actin binding motif by examining the enzymatic, motile, and cross-linking properties of full length myosin IIIB and myosin IIIA, with and without a functional tail actin binding motif. We will also examine the structural basis for the interaction of the tail actin binding motif with actin filaments. The movement of myosin IIIA along actin bundles will be examined with single molecule motility assays. The biochemically characterized constructs will be examined in cultured HeLa/COS cells, photoreceptors, and inner ear hair cells to determine how the activity of myosin III mediates its cellular localization, actin dynamics, and in vivo motility. Overall, studying the enzymatic, motor, and regulatory properties of myosin IIIA and IIIB in vitro and in cell biology experiments will identify its physiological role in sensory cells. In addition, these studies well provide the basis for understanding how disruption of the myosin IIIA gene leads to deafness and retinal degeneration.

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III的酶和运动特性，使其在感觉细胞中发挥关键作用。肌球蛋白IIIA基因的破坏导致脊椎动物耳聋以及无脊椎动物视网膜变性和光转导缺陷。肌球蛋白III是一种基于肌动蛋白的分子马达，包含肌球蛋白超家族特有的保守马达结构域，但也包含N-末端激酶结构域。我们提出激酶活性是通过激酶结构域激活环的磷酸化激活的，其刺激马达结构域中的环2的自磷酸化并导致马达活性下调。磷酸酶活性使马达返回其活性状态并使激酶结构域失活。我们将研究肌球蛋白IIIA结构的酶，运动和结构特性与激酶结构域激活/失活，以及电机结构域激活/失活。肌球蛋白IIIA与肌球蛋白IIIB不同之处在于其C-末端尾部含有肌动蛋白结合基序。我们建议，额外的肌动蛋白结合位点的尾巴允许肌球蛋白IIIA交联肌动蛋白丝，协同激活电机酶循环，并通过增加肌球蛋白IIIA的肌动蛋白的整体亲和力，提高其能动性。我们将直接确定尾部肌动蛋白结合基序的作用，通过检查全长肌球蛋白IIIB和肌球蛋白IIIA的酶，运动和交联特性，有和没有一个功能性的尾部肌动蛋白结合基序。我们还将研究尾部肌动蛋白结合基序与肌动蛋白丝相互作用的结构基础。肌球蛋白IIIA沿着肌动蛋白束的运动将采用单分子运动试验进行检查。将在培养的HeLa/COS细胞，光感受器和内耳毛细胞中检查生物化学表征的构建体，以确定肌球蛋白III的活性如何介导其细胞定位，肌动蛋白动力学和体内运动性。总的来说，研究肌球蛋白IIIA和IIIB在体外和细胞生物学实验中的酶促，运动和调节特性将确定其在感觉细胞中的生理作用。此外，这些研究为理解肌球蛋白IIIA基因的破坏如何导致耳聋和视网膜变性提供了基础。