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：
8129560
负责人：
CHRISTOPHER M YENGO
金额：
$ 25.8万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
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 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8129560
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 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 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）：该研究项目的长期目标是了解肌球蛋白III的酶促和运动性能，使其能够在感觉细胞中起关键作用。肌球蛋白IIIA基因的破坏会导致脊椎动物的耳聋，视网膜退化和无脊椎动物的光转导缺陷。肌球蛋白III是一种基于肌动蛋白的分子运动，它包含肌球蛋白超家族的保守运动结构域特征，但还包含N末端激酶结构域。我们提出，激酶活性通过激酶结构域激活环的磷酸化激活，该激酶活化环刺激运动域中环2的自磷酸化并导致运动活性下调。磷酸酶活性将电动机恢复到其活性状态，并使激酶结构域失活。我们将检查具有激酶域激活/失活的肌球蛋白IIIA构建体的酶促，运动和结构特性，以及运动结构域激活/失活。肌球蛋白IIIA与肌球蛋白IIIB不同，因为它在其C末端尾部包含肌动蛋白结合基序。我们建议，尾部中的其他肌动蛋白结合位点使肌球蛋白IIIA可以交叉肌动蛋白丝，合作激活运动酶循环，并通过增加肌球蛋白IIIA对肌动蛋白的整体亲和力来增强其运动性能。我们将通过检查全长肌球蛋白IIIB和肌球蛋白IIIA的酶促，运动和交联的特性，直接确定尾肌动蛋白结合基序的作用，并具有和不具有功能性尾巴肌动蛋白结合基序。我们还将研究尾肌蛋白结合基序与肌动蛋白丝的相互作用的结构基础。肌球蛋白IIIA沿肌动蛋白束的运动将通过单分子运动测定法检查。生化特征的构建体将在培养的HeLa/cos细胞，感光细胞和内耳毛细胞中进行检查，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。总体而言，在体外和细胞生物学实验中研究肌球蛋白IIIA和IIIB的酶促，运动和调节性能将确定其在感觉细胞中的生理作用。此外，这些研究很好地为理解肌球蛋白IIIA基因的破坏如何导致耳聋和视网膜变性提供了基础。