Exploring the unexpected roles of the sarcomeric myosin family member MYH7b

探索肌节肌球蛋白家族成员 MYH7b 的意想不到的作用

基本信息

批准号：
10203913
负责人：
Lindsey Anne Lee
金额：
$ 3.42万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2022-05-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10203913
关键词：
Actins Address Anatomy Animals Auditory Auditory system Behavioral Biochemical Biological Biophysics Birds Brain Clustered Regularly Interspaced Short Palindromic Repeats Complement Defect Detection Disease Environment Evolution Family Family member Foundations Gene Expression Gene Family Genes Goals Health Hearing Higher Order Chromatin Structure Human In Vitro Knockout Mice Labyrinth Mammals Molecular Motor Motor Activity Mus Muscle Muscle Contraction Muscle Spindles Muscle function Mutation Myocardium Myosin ATPase Myosin Heavy Chains Phenotype Physiological Process Property Protein Isoforms Proteins Pythons Recombinant Proteins Recombinants Reptiles Rod Role Sarcomeres Skeletal Muscle Snakes Striated Muscles Structure Tissues Vertebrates Whole Organism Wild Type Mouse Work biophysical properties experimental study hearing impairment hereditary hearing loss insight member mouse model novel orbit muscle

项目摘要

PROJECT SUMMARY The sarcomeric myosins responsible for muscle contraction are among the most well studied proteins, yet we are still discovering new roles for this class of molecule. Myosin heavy chain 7b (MYH7b) is a recently identified gene belonging to the sarcomeric myosin family that appears to have evolved new roles in mammalian non-muscle tissue. In mammals, MYH7b protein is only expressed in specialized tissues such as extraocular muscle and muscle spindles and, surprisingly, is found in the inner ear and brain. These observations are highly unusual as no other muscle myosin has ever been found in non-sarcomeric tissues. Further, mutations in MYH7b cause hearing loss in humans with no apparent muscle phenotypes, indicating that MYH7b has an important yet undefined role in non-muscle tissue. Intriguingly, in snakes, MYH7b is found in the sarcomeres of cardiac and skeletal muscles. Despite these divergent roles in reptiles and mammals, MYH7b shares high sequence identity between these classes of vertebrates, so it is unclear how mammalian MYH7b function may deviate in non-muscle tissues from its expected sarcomeric role. The goal of this proposal is to identify the role and activity of MYH7b in mammalian non-muscle tissue and understand how mutations in MYH7b contribute to hearing loss. I hypothesize that distinct biophysical and biochemical properties allow mammalian MYH7b to function in non-muscle environments, and that mutations in MYH7b disrupt hearing by causing this protein to interfere with normal auditory processes. In Aim 1, I will determine the physiological role of MYH7b in mammals using mouse models. First, I will investigate the role of MYH7b in wild type mice by determining subcellular localization, protein interactions, and structures formed by MYH7b in the inner ear. Next, I will investigate any anatomical and behavioral defects in MYH7b null mice that our lab is currently maintaining. Finally, I will use CRISPR/Cas gene editing to introduce the human MYH7b mutations that cause hearing loss into mice and assess hearing function and any resultant anatomical or behavioral defects. I anticipate this work will reveal how MYH7b functions in the auditory system, which will provide a foundation for understanding MYH7b function in the mammalian brain. In Aim 2, I will investigate the molecular function of MYH7b by comparing the biophysical and biochemical activities of recombinant python and human MYH7b protein. These analyses will address the question of whether these proteins have similar molecular functions, or whether the mammalian MYH7b activity has diverged from other sarcomeric myosins. I will also determine the functional impact of the two hearing loss mutations (one in the catalytic motor domain and one in the structural rod domain of the myosin) on motor properties and the molecule's ability to self assemble into ordered structures. Collectively, this work will provide a comprehensive understanding of the role of mammalian MYH7b and its activity in non-muscle tissue at the molecular, cellular, and whole organism level as well as provide a foundation for a better understanding of MYH7b evolution and divergence across species.

项目摘要负责肌肉收缩的肉瘤肌球蛋白是研究最精心良好的蛋白质之一，但是我们仍在发现这类分子的新角色。肌球蛋白重链7B（MYH7B）是最近的鉴定出属于肉瘤肌球蛋白家族的基因，似乎在哺乳动物非肌肉组织。在哺乳动物中，MYH7B蛋白仅在专业组织中表达眼外肌肉和肌肉纺锤体，令人惊讶地在内耳和大脑中发现。这些观察结果是高度不寻常的，因为在非肉瘤组织中没有发现其他肌肉肌球蛋白。此外，MYH7B的突变会导致人类的听力损失，没有明显的肌肉表型，表明 MYH7B在非肌肉组织中具有重要但不确定的作用。有趣的是，在蛇中发现了myh7b 在心脏和骨骼肌的肉瘤中。尽管在爬行动物和哺乳动物中这些发散作用，但 MYH7B在这些类别的脊椎动物之间具有很高的序列身份，因此目前尚不清楚哺乳动物如何 MYH7B功能可能在非肌肉组织中偏离其预期的肌肉作用。目标的目标建议是确定MYH7B在哺乳动物非肌肉组织中的作用和活动，并了解如何 MYH7B中的突变导致听力损失。我假设这种独特的生物物理和生化属性允许哺乳动物MYH7B在非肌肉环境中起作用，而MYH7B中的突变通过导致这种蛋白质干扰正常听觉过程，破坏听力。在AIM 1中，我将确定 MYH7B在哺乳动物中使用小鼠模型的生理作用。首先，我将调查MYH7B在野外的作用通过确定MYH7B在中的亚细胞定位，蛋白质相互作用以及在内耳。接下来，我将研究MYH7B无效老鼠中的任何解剖和行为缺陷，我们的实验室是目前维护。最后，我将使用CRISPR/CAS基因编辑来介绍人类MYH7B突变这会导致听力丧失对小鼠的损失并评估听力功能以及任何结果的解剖或行为缺陷。我预计这项工作将揭示MYH7B在听觉系统中的功能，这将提供一个理解哺乳动物大脑中MYH7B功能的基础。在AIM 2中，我将研究分子通过比较重组Python和人类的生物物理和生化活性，MYH7B的功能 myh7b蛋白。这些分析将解决这些蛋白是否具有相似分子的问题功能，或哺乳动物MYH7B活性是否与其他肉类肌球蛋白有所不同。我也会确定两个听力损失突变的功能影响（一个在催化运动结构域中，一个在肌球蛋白的结构杆结构域）在运动性能和分子自我组装的能力上有序结构。总的来说，这项工作将对哺乳动物MYH7B及其在分子，细胞和整个生物水平的非肌肉组织中的活性并为更好地理解MYH7B的进化和跨物种的分歧提供了基础。