Exploring the unexpected roles of the sarcomeric myosin family member MYH7b

项目总结 负责肌肉收缩的肌球蛋白是研究最充分的蛋白质之一，然而我们 仍在为这类分子发现新的角色。肌球蛋白重链7b(MYH7b)是最近发现的 鉴定的属于肌节肌球蛋白家族的基因似乎已经进化出新的角色 哺乳动物的非肌肉组织。在哺乳动物中，MYH7b蛋白只在特定的组织中表达，如 眼外肌和肌梭，令人惊讶的是，发现在内耳和大脑。这些 观察结果非常不寻常，因为在非肌瘤组织中从未发现过其他肌肉肌球蛋白。 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在非肌肉组织中有一个重要但尚未确定的作用。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扮演着不同的角色， MYH7b在这些脊椎动物之间有很高的序列同源性，所以还不清楚哺乳动物 MYH7b在非肌肉组织中的功能可能偏离其预期的肌节作用。这样做的目的是 建议确定MYH7b在哺乳动物非肌肉组织中的作用和活性，并了解如何 MYH7b基因突变会导致听力损失。我假设不同的生物物理和生化 特性允许哺乳动物MYH7b在非肌肉环境中发挥作用，MYH7b的突变 通过使这种蛋白质干扰正常的听觉过程来扰乱听力。在目标1中，我将确定 MYH7b在哺乳动物小鼠模型中的生理作用。首先，我将研究MYH7b在野生型中的作用 通过确定MYH7b在小鼠体内的亚细胞定位、蛋白质相互作用和形成的结构来确定小鼠的类型 内耳。接下来，我将调查我们实验室MYH7b基因缺失小鼠的任何解剖和行为缺陷 目前正在维护。最后，我将使用CRISPR/Cas基因编辑来介绍人类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如何在听觉系统中发挥作用，这将提供一种 为了解MYH7b在哺乳动物脑中的功能奠定基础。在目标2中，我将研究分子 通过比较重组蟒蛇和人的生物物理和生化活性来研究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。 或者哺乳动物的MYH7b活性是否与其他肌节肌球蛋白不同。我也会 确定两个听力损失突变(一个在催化运动区，一个在 肌球蛋白的结构杆状结构域)对运动特性和分子自组装成 有序的结构。总体而言，这项工作将使我们全面了解 哺乳动物MYH7b及其在非肌肉组织中的分子、细胞和整体水平的活性 并为更好地理解MYH7b的进化和物种间的差异提供了基础。

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.