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

• 批准号: 10666442
• 负责人: Brett A Colson
• 金额: $ 44.24万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 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/10666442
• 关键词: Acceleration; Actins; Actomyosin; Address; Affect; Anisotropy; Arthrogryposis; Artificial skin; Binding; Biological Assay; Cardiac Myosins; Contracts; Contracture; Cyclic AMP-Dependent Protein Kinases; Deceleration; 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; Permeability; Phosphoproteins; Phosphorylation; Physiological; Positioning Attribute; Post-Translational Protein Processing; Protein Dynamics; Protein Isoforms; Proteins; Publishing; RNA Splicing; Recombinants; Regulation; Reporter; Reporting; Research; Resolution; Role; Sampling; Sarcomeres; Site; Skeletal Muscle; Slide; Stains; Structure; Structure-Activity Relationship; Testing; Therapeutic; Thick Filament; Thin Filament; Time; Tropomyosin; Upper Extremity; Variant; Visualization; Work; 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; time use; tool

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.

项目总结 每3,000名活产儿中就有1人患有关节畸形，导致上肢和下肢关节痉挛。 没有治愈方法，这使它成为一种未得到满足的医疗需求。编码慢速骨骼的MYBPC1基因突变 在慢肌和快肌中表达的肌球蛋白结合蛋白C(sMyBP-C)与 远端关节紊乱病(DA)。MYBPC2编码快速骨架MyBP-C(fMyBP-C)，仅在FAST-C中发现 肌肉抽搐。作为一种肌球蛋白锚定的肌肉蛋白，MyBP-C向肌动蛋白延伸，定位于 肌节调节肌球蛋白在力量发育中的相互作用。骨骼肌中的MyBP-C有三种 主要调控因子：异构体(慢与快)、剪接变异体(长与短的sMyBP-C)和翻译后 修饰(磷酸化)。SMyBP-C在其N端被蛋白激酶A(PKA)磷酸化。这个 SMyBP-C的作用(S)、其在骨骼肌中的磷酸化和DA突变尚不清楚。我们的预赛 对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 已经开发出创新的生物物理工具，能够评估骨骼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 证明我们已经成功地在N端sMyBP-C中开发了荧光传感器，其结构 动力学对PKA介导的磷酸化和与肌动蛋白的结合非常敏感。我们还开发了 解决fMyBP-C、long sMyBP-C和Short之间肌动蛋白结合的分子间荧光分析 MyBP-C由于磷酸化和肌动蛋白上的原肌球蛋白的存在。这些初步结果表明 对不同骨骼MyBP-C的关键生理调节机制，并提供了更多的科学依据 进行拟议研究的前提和可行性。目的1将评估sMyBP-C结合和 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， 肌动蛋白和肌球蛋白。光谱探针将被放置在这些蛋白质中，并将采用各种方法来 利用野生型和DA突变体sMyBP-C在体外和原位检测关键构象。对于光纤实验， 将从新的sMyBP-C基因敲除(KO)小鼠中分离肌肉，并用重组sMyBP- C、DA突变体和肌肉蛋白探针。将评估样本的结合和收缩功能。目标 2将确定PKA介导的sMyBP-C磷酸化如何影响AIM 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，但使用fMyBP-C KO和 SMyBP-C/fMyBP-C双KO小鼠进行纤维实验。拟议的研究捕捉到了结构动力学 以及使用新的高分辨率方法在实时和肌丝空间中进行交互。这些目标 概述在收缩周期中研究正常和突变骨骼MyBP-C的逐步计划。通过 监测蛋白质上的点之间的距离以及这些距离的顺序(或无序) 在生理条件下，突变体可以被分到垃圾箱中，以便于进行基于机械的靶向治疗。