Cut and paste of myosin binding protein-C in skeletal muscles

骨骼肌中肌球蛋白结合蛋白-C 的剪切和粘贴

基本信息

批准号：
10571115
负责人：
Samantha P Harris
金额：
$ 20.26万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-10 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10571115
关键词：
Acute Affect Alternative Splicing Arthrogryposis Cardiac Cardiac Myocytes Cardiac Myosins Complex Congenital clubfoot Consensus Contracture Data Data Analyses Defect Detergents Development Disease Distal Excision Familial Tremors Family Fiber Functional disorder Genes Genetic Engineering Goals Hypertrophic Cardiomyopathy In Situ In Vitro Knock-out Knockout Mice Link Methods Modeling Modification Mus Muscle Muscle Cells Muscle Contraction Muscle Fibers Muscle Tremors Muscle function Mutation Myocardium Myopathy N-terminal Paste substance Permeability Persons Phosphorylation Post-Translational Protein Processing Protein Splicing Proteins RNA Splicing Recombinant Proteins Recombinants Relaxation Reporting Role Sarcomeres Site Skeletal Muscle Syndrome TEV protease Time Tremor Up-Regulation Variant Work covalent bond differential expression disease-causing mutation gene product genetic regulatory protein insight mouse model myosin-binding protein C novel novel strategies paralogous gene prevent skeletal

项目摘要

PROJECT SUMMARY The overall goal of this project is to understand how myosin binding protein-C (MyBP-C) regulates contraction and relaxation in skeletal muscles. 3 distinct paralogs of MyBP-C encoded by 3 separate genes, MYBPC1, MYBPC2, and MYBPC3, are expressed in slow twitch skeletal muscle, fast twitch muscle, and cardiac muscle, respectively. Of the three, the cardiac paralog has been most intensively studied because mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). However, like MYBPC3, it is now clear that mutations in MYBPC1 and MYBPC2 are increasingly linked to congenital skeletal muscle diseases such as distal arthrogryposis and more recently to a new class of familial muscle tremors. Despite this, it has been challenging to disentangle the distinct functional roles of the 2 skeletal paralogs of MyBP-C in part because most skeletal muscles contain a mixture of slow and fast fiber types and because MyBP-C1, expressed in slow twitch fibers, undergoes extensive alternative splicing resulting in a family of differentially expressed proteins each with unique functional effects. As a result, it has been nearly impossible to distinguish the functional significance of each variant in the context of working muscles. To overcome these challenges, the PI’s lab recently developed a novel “cut and paste” approach to selectively target and replace different MyBP-C paralogs in muscle sarcomeres. The method, first developed for MYBPC3, relies on the use of gene-edited mice that express a tobacco etch virus protease (TEVp) consensus site and a “SpyTag” sequence within cardiac MyBP-C so that when detergent-permeabilized myocytes are treated with TEVp MyBP-C is selectively cleaved. Next, MyBP-C can be replaced with any recombinant protein (containing any desired sequence modification) as long as the recombinant protein encodes a “SpyCatcher” sequence because SpyCatcher and SpyTag from an instantaneous covalent bond. Here, we expanded the method by creating “SpyC1” mice that allow us to selectively study slow skeletal MyBP-C (Aim 1) and “SnoopC2” mice to study fast skeletal MyBP-C (Aim 2). Preliminary data using the cut and paste method has already yielded new insights into how MyBP-C dysfunction can lead to muscle tremors and additional results from these studies will determine the functional significance of each MyBP-C paralog and how mutations in MyBP-C cause disease.

项目摘要该项目的总体目标是了解肌球蛋白结合蛋白-C（MYBP-C）如何调节骨骼肌的收缩和放松。 3个不同的MYBP-C的旁系同源物，由3个单独编码基因，mybpc1，mybpc2和mybpc3以缓慢的抽搐骨骼肌肉表示，快速抽搐肌肉和心肌。在这三个中，心脏旁系同源物是最强烈的研究了，因为MYBPC3中的突变是肥厚性心肌病的最常见原因（HCM）。但是，像MYBPC3一样，现在很明显MyBPC1和MyBPC2中的突变是越来越多地与先天性骨骼肌疾病有关最近进入新的家庭肌肉震颤。尽管如此，解开态一直是挑战 MYBP-C的两个骨骼旁系同源物的独特功能作用部分是因为大多数骨骼肌肉包含缓慢和快速纤维类型的混合物，并且由于用缓慢的抽搐纤维表示MYBP-C1，因此经历广泛的替代剪接，导致一个不同表达蛋白的家族具有独特的功能效果。结果，几乎不可能区分功能在工作肌肉的背景下，每个变体的重要性。为了克服这些挑战，PI的挑战实验室最近开发了一种新颖的“切割和糊状”方法，以选择性地靶向和替换不同 MYBP-C肌肉肉瘤中的旁系同源物。该方法最初是为MyBPC3开发的，它依赖于使用表达烟草蚀刻病毒蛋白酶（TEVP）共识位点和“ spytag”的基因编辑的小鼠心脏mybp-c中的序列，因此，确定渗透性的心肌细胞时会用 TEVP MYBP-C有选择地分裂。接下来，可以用任何重组蛋白代替MYBP-C （包含任何所需序列修饰），只要重组蛋白编码A “间谍捕获者”序列是因为瞬时共价键的间谍捕获器和间谍序列。这里，我们通过创建“ spyc1”小鼠扩展了该方法，使我们能够选择性地研究慢速骨骼 MYBP-C（AIM 1）和“ Snoopc2”小鼠研究快速骨骼MYBP-C（AIM 2）。使用初步数据剪切和糊状方法已经对MYBP-C功能障碍如何导致的新见解产生了新的见解肌肉震颤和这些研究的其他结果将决定每个MYBP-C旁系同源物以及MYBP-C中的突变如何引起疾病。