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Skeletal Myosin-Binding Protein C Regulation and Structural Dynamics

骨骼肌球蛋白结合蛋白 C 调节和结构动力学

基本信息

批准号：
10442876
负责人：
Brett A Colson
金额：
$ 45.7万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10442876
关键词：
Actins Actomyosin Address Affect Anisotropy Arthrogryposis Artificial skin Binding Biological Assay Cardiac Myosins Contracts Contracture Cyclic AMP-Dependent Protein Kinases Development Disease Distal Equilibrium Evaluation Fiber Fluorescence Fluorescence Resonance Energy Transfer Frequencies Gel Genes Health Human Hypertrophic Cardiomyopathy In Situ In Vitro Joints Kinetics Knock-out Knockout Mice Knowledge Label Length Live Birth Lower Extremity Mass Spectrum Analysis Measures Mediating Medical Microfilaments Molecular Molecular Conformation Monitor Mus Muscle Muscle Cells Muscle Fibers Muscle Proteins Muscle Weakness Muscle function Mutant Strains Mice Mutation Myocardial Myopathy Myosin ATPase N-terminal Nonmuscle Myosin Type IIA Outcome Pathogenicity Performance Phosphoproteins Phosphorylation Physiological Positioning Attribute Post-Translational Protein Processing Protein Isoforms Proteins RNA Splicing Recombinants Regulation Reporter Reporting Research Resolution Role Sampling Sarcomeres Site Skeletal Muscle Stains Structure Structure-Activity Relationship Testing Therapeutic Thick Filament Thin Filament Time Tropomyosin Upper Extremity Variant Work base biophysical tools design effective therapy experimental study improved in vivo innovation mouse model mutant myosin-binding protein C novel novel therapeutics phosphorescence prevent protein structure sensor skeletal spectroscopic data tool

项目摘要

PROJECT SUMMARY Arthrogryposis is present in 1 in 3,000 live births causing joint contractures in both upper and lower limbs. There is no cure making it an unmet medical need. Mutations in the MYBPC1 gene encoding slow skeletal myosin-binding protein C (sMyBP-C), expressed in both slow and fast muscle types are associated with distal arthrogryposis (DA). MYBPC2 encodes for fast skeletal MyBP-C (fMyBP-C) and is found only in fast- twitch muscle. As a myosin-anchored protein of muscle, MyBP-C extends toward actin, positioned centrally in the sarcomere to regulate actomyosin interactions in force development. MyBP-C in skeletal muscle has three major regulators: isoform (slow vs. fast), splice variant (long vs. short sMyBP-C), and posttranslational modification (phosphorylation). sMyBP-C is phosphorylated by protein kinase A (PKA) at its N terminus. The role(s) of sMyBP-C, its phosphorylation and DA mutations in skeletal muscle are not known. Our preliminary studies of sMyBP-C show that binding to actomyosin is dependent on phosphorylation and DA mutations. We have developed innovative biophysical tools that enable evaluation of skeletal MyBP-C structural dynamics, actomyosin interactions in muscle, and effects of phosphorylation and mutations. Our new preliminary studies demonstrate that we have successfully developed fluorescent sensors in N terminal sMyBP-C whose structure and dynamics are sensitive to PKA-mediated phosphorylation and binding to actin. We have also developed inter-molecular fluorescence assays that resolve actin binding between fMyBP-C, long sMyBP-C, and short MyBP-C due to phosphorylation and the presence of tropomyosin on actin. These preliminary results suggest key physiological mechanisms of regulation for the different skeletal MyBP-C and provides additional scientific premise and feasibility for pursuing the proposed studies. Aim 1 will evaluate effects of sMyBP-C binding and DA mutations on interactions with actomyosin, capturing structure and proximities of N terminal sMyBP-C, actin and myosin. Spectroscopic probes will be placed in these proteins and approaches will be employed to detect key conformations in vitro and in situ with wild type and DA mutant sMyBP-C. For fiber experiments, muscle will be isolated from novel sMyBP-C knockout (KO) mice and permeabilized with recombinant sMyBP- C, DA mutants, and muscle protein probes. Samples will be assessed for binding and contractile function. Aim 2 will determine how PKA-mediated phosphorylation of sMyBP-C affects the parameters evaluated in Aim 1. Aim 3 will determine how fMyBP-C affects the parameters evaluated in Aim 1 except using fMyBP-C KO and sMyBP-C/fMyBP-C double-KO mice for fibers experiments. The proposed studies capture structural dynamics and interactions in real time and myofilament space using novel high-resolution approaches. These aims outline a stepwise plan for studying normal and mutant skeletal MyBP-C during the contractile cycle. By monitoring distances between points on proteins and the order (or disorder) of those distances under physiological conditions, mutants can be separated into bins to facilitate targeted mechanistic-based therapies.

项目概要每 3,000 名活产儿中就有 1 人患有关节弯曲，导致上肢和下肢关节挛缩。目前尚无治愈方法，使其成为未得到满足的医疗需求。编码慢骨骼的 MYBPC1 基因突变肌球蛋白结合蛋白 C (sMyBP-C) 在慢肌和快肌类型中表达，与远端关节弯曲（DA）。 MYBPC2 编码快速骨骼 MyBP-C (fMyBP-C)，仅在快速骨骼中发现。抽动肌肉。作为肌肉的肌球蛋白锚定蛋白，MyBP-C 延伸至肌动蛋白，位于肌动蛋白的中心位置肌节调节力量发展中肌动球蛋白的相互作用。骨骼肌中的 MyBP-C 有 3 个主要调节因子：亚型（慢与快）、剪接变体（长与短 sMyBP-C）和翻译后修饰（磷酸化）。 sMyBP-C 的 N 末端被蛋白激酶 A (PKA) 磷酸化。这 sMyBP-C 的作用、其磷酸化和 DA 突变在骨骼肌中的作用尚不清楚。我们的初步 sMyBP-C 的研究表明，与肌动球蛋白的结合依赖于磷酸化和 DA 突变。我们开发了创新的生物物理工具，可以评估骨骼 MyBP-C 结构动力学，肌动球蛋白在肌肉中的相互作用，以及磷酸化和突变的影响。我们新的初步研究证明我们已经成功开发出N端荧光传感器sMyBP-C，其结构动力学对 PKA 介导的磷酸化和与肌动蛋白的结合敏感。我们还开发了分子间荧光测定可解析 fMyBP-C、长 sMyBP-C 和短 sMyBP-C 之间的肌动蛋白结合 MyBP-C 由于磷酸化和肌动蛋白上原肌球蛋白的存在而产生。这些初步结果表明不同骨骼 MyBP-C 调节的关键生理机制，并提供了额外的科学依据进行拟议研究的前提和可行性。目标 1 将评估 sMyBP-C 结合的效果和与肌动球蛋白相互作用的 DA 突变，捕获 N 末端 sMyBP-C 的结构和邻近性，肌动蛋白和肌球蛋白。将在这些蛋白质中放置光谱探针，并采用方法来使用野生型和 DA 突变体 sMyBP-C 体外和原位检测关键构象。对于纤维实验，肌肉将从新型 sMyBP-C 敲除 (KO) 小鼠中分离出来，并用重组 sMyBP- 进行透化 C、DA 突变体和肌肉蛋白探针。将评估样品的结合和收缩功能。目的 2 将确定 PKA 介导的 sMyBP-C 磷酸化如何影响目标 1 中评估的参数。目标 3 将确定 fMyBP-C 如何影响目标 1 中评估的参数，除了使用 fMyBP-C KO 和 sMyBP-C/fMyBP-C 双 KO 小鼠用于纤维实验。拟议的研究捕捉结构动力学使用新颖的高分辨率方法进行实时和肌丝空间的交互。这些目标概述了研究收缩周期中正常和突变骨骼 MyBP-C 的逐步计划。经过监测蛋白质上点之间的距离以及这些距离的顺序（或无序）根据生理条件，突变体可以分为不同的箱，以促进基于机制的靶向治疗。