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

• 批准号: 9760706
• 负责人: Lindsey Anne Lee
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760706
• 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; 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; retinal rods

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.

项目概要 负责肌肉收缩的肌节肌球蛋白是研究最深入的蛋白质之一，但我们 仍在发现此类分子的新作用。肌球蛋白重链 7b (MYH7b) 是最近 鉴定出属于肌节肌球蛋白家族的基因，该基因似乎在 哺乳动物的非肌肉组织。在哺乳动物中，MYH7b 蛋白仅在特殊组织中表达，例如 令人惊讶的是，眼外肌和肌梭也存在于内耳和大脑中。这些 观察结果非常不寻常，因为在非肌节组织中尚未发现其他肌肉肌球蛋白。 此外，MYH7b 的突变会导致没有明显肌肉表型的人类听力损失，这表明 MYH7b 在非肌肉组织中具有重要但尚未明确的作用。有趣的是，在蛇中发现了 MYH7b 在心肌和骨骼肌的肌节中。尽管爬行动物和哺乳动物的作用不同， MYH7b 在这些脊椎动物类别之间具有高度的序列同一性，因此尚不清楚哺乳动物如何 MYH7b 功能在非肌肉组织中可能偏离其预期的肌节作用。此举的目标 提议是确定 MYH7b 在哺乳动物非肌肉组织中的作用和活性，并了解如何 MYH7b 突变会导致听力损失。我假设不同的生物物理和生物化学 特性允许哺乳动物 MYH7b 在非肌肉环境中发挥作用，并且 MYH7b 中的突变 通过使这种蛋白质干扰正常的听觉过程来破坏听力。在目标 1 中，我将确定 使用小鼠模型研究 MYH7b 在哺乳动物中的生理作用。首先，我将研究 MYH7b 在野生环境中的作用 通过确定亚细胞定位、蛋白质相互作用以及 MYH7b 在小鼠体内形成的结构来识别小鼠类型 内耳。接下来，我将调查我们实验室的 MYH7b 缺失小鼠的任何解剖学和行为缺陷 目前维护中。最后，我将使用CRISPR/Cas基因编辑来引入人类MYH7b突变 导致小鼠听力损失并评估听力功能和任何由此产生的解剖或行为 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 为理解哺乳动物大脑中的 MYH7b 功能奠定了基础。在目标 2 中，我将研究分子 通过比较重组蟒蛇和人类的生物物理和生化活性来了解 MYH7b 的功能 MYH7b 蛋白。这些分析将解决这些蛋白质是否具有相似分子的问题 功能，或哺乳动物 MYH7b 活性是否与其他肌节肌球蛋白不同。我也会 确定两种听力损失突变（一种在催化运动域，一种在 肌球蛋白的结构杆域）对运动特性和分子自组装能力的影响 有序的结构。总的来说，这项工作将提供对以下角色的全面理解： 哺乳动物 MYH7b 及其在分子、细胞和整个生物体水平的非肌肉组织中的活性 并为更好地了解 MYH7b 进化和跨物种分化奠定基础。