Functional diversity of myosin VII

肌球蛋白 VII 的功能多样性

基本信息

批准号：
7932424
负责人：
ENRIQUE M DE LA CRUZ
金额：
$ 4.24万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7932424
关键词：
ADP-G-actin ATP phosphohydrolase Actins Actomyosin Adhesions Affect Affinity Amino Acids Anisotropy Behavior Binding Binding Sites Biochemical Biological Biological Assay Biological Process Brush Border Cells Chemicals Chimera organism Chimeric Proteins Coiled-Coil Domain Data Dependence Dimerization Disease Engineering Equilibrium Filament Functional disorder Generations Head Human In Vitro Investigation Kidney Kinetics Labyrinth Length Link MYO7A gene Measurement Measures Mediating Membrane Membrane Proteins Methods Microfilaments Molecular Molecular Conformation Motor Motor Activity Muscle Rigidity Mutation Myosin ATPase Myosin Type V Nucleotides Organism Phenotype Play Point Mutation Population Property Protein Isoforms Proteins Reaction Research Personnel Resolution Retina Role Side Slide Structure Techniques Testing Usher Syndrome Variant analytical ultracentrifugation base cell motility cellular microvillus clinically relevant deafness density design dimer hearing impairment human disease insight intestinal epithelium member monomer mutant myosin VI particle phosphorescence physical property research study sedimentation equilibrium time use trafficking

项目摘要

DESCRIPTION (provided by applicant): Humans have two class VII myosins, myosin VIIa and VIIb, which are very similar in domain structure except that myosin VIIb possesses a unique 19 amino acid insert at the actin-binding site and lacks a putative coiled-coil domain. Mutations in myosin VIIa have been linked to type 1b Usher syndrome, the most common deafness-blindness disorder in humans, and two other hearing loss disorders in humans, DFNB2 and DFNA11. Myosin VIIb is believed to traffic and organize membranes and membrane-associated proteins. Despite the critical roles class VII myosins play in cells, understanding the basis of myosin VII function and the deafness phenotype associated with myosin VIIa has been limited by a lack of information regarding the chemical and physical properties of myosin VII. Efforts in this proposal focus on understanding the enzymatic and motor properties of myosin VIIa and VIIb. Aim 1 will determine if full-length myosin VIIa and VIIb are monomers or dimers. Aim 2 will provide a complete kinetic characterization of the myosin VIIa and VIIb ATPase cycles, determine the contributions of the 19 amino acid insertion to the ATPase cycle kinetics through the generation of mutants and chimeric proteins, and identify how mutations that generate deafness affect myosin VIIa motor activity. Aim 3 will measure how external loads modulate ADP and actin binding to high duty ratio myosins and evaluate mechanisms of strain-dependent head-head coordination. Aim 4 will employ high-resolution spectroscopic methods to measure cooperative changes in the structural dynamics of actin filaments induced by high duty ratio myosin binding. The two highly conserved isoforms provide a unique opportunity to test the structural and functional effects of the actin-binding loop and of dimerization on myosin function. The proposed detailed analysis of reaction mechanisms and structural dynamics will help develop testable hypotheses regarding the function and mechanism of myosin VII, and illustrate how sequence and structural variations in myosin motors define their enzymatic properties, which ultimately dictates their biological functions. Because mutations and disruption of myosin VIIa function are of clinical relevance, the proposed experiments will advance our understanding of the molecular basis of several human diseases.

描述（由申请人提供）：人类有两种 VII 类肌球蛋白，肌球蛋白 VIIa 和 VIIb，它们在结构域结构上非常相似，只是肌球蛋白 VIIb 在肌动蛋白结合位点具有独特的 19 个氨基酸插入并且缺乏假定的卷曲螺旋领域。肌球蛋白 VIIa 的突变与 1b 型 Usher 综合征（人类最常见的耳聋失明性疾病）以及另外两种人类听力损失疾病 DFNB2 和 DFNA11 有关。肌球蛋白 VIIb 被认为负责运输和组织膜和膜相关蛋白。尽管 VII 类肌球蛋白在细胞中发挥着关键作用，但由于缺乏有关肌球蛋白 VII 的化学和物理特性的信息，对肌球蛋白 VII 功能的基础和与肌球蛋白 VIIa 相关的耳聋表型的了解受到限制。该提案的重点是了解肌球蛋白 VIIa 和 VIIb 的酶学和运动特性。目标 1 将确定全长肌球蛋白 VIIa 和 VIIb 是单体还是二聚体。目标 2 将提供肌球蛋白 VIIa 和 VIIb ATP 酶循环的完整动力学表征，通过突变体和嵌合蛋白的生成确定 19 个氨基酸插入对 ATP 酶循环动力学的贡献，并确定产生耳聋的突变如何影响肌球蛋白 VIIa运动活动。目标 3 将测量外部负载如何调节 ADP 和肌动蛋白与高占空比肌球蛋白的结合，并评估应变依赖性头-头协调机制。目标 4 将采用高分辨率光谱方法来测量由高占空比肌球蛋白结合引起的肌动蛋白丝结构动力学的协同变化。这两种高度保守的亚型提供了独特的机会来测试肌动蛋白结合环和二聚化对肌球蛋白功能的结构和功能影响。所提出的对反应机制和结构动力学的详细分析将有助于开发关于肌球蛋白 VII 的功能和机制的可测试假设，并说明肌球蛋白马达的序列和结构变化如何定义其酶特性，从而最终决定其生物学功能。由于肌球蛋白 VIIa 功能的突变和破坏具有临床相关性，因此拟议的实验将增进我们对几种人类疾病的分子基础的理解。