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: 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
• 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

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.

项目摘要 关节炎存在于3,000名活出生中的1个，在上肢和下肢引起联合染色。 无法治愈，这是未满足的医疗需求。编码缓慢骨骼的MyBPC1基因中的突变 肌球蛋白结合蛋白C（SMYBP-C）在慢速和快速肌肉类型中表达都与 远端关节炎（DA）。 MYBPC2编码快速骨骼MYBP-C（FMYBP-C），仅在快速中发现 抽搐肌肉。作为肌球蛋白锚定的肌肉的蛋白质，MyBP-C向肌动蛋白延伸，位于集中位于 肌肉肌球蛋白相互作用在实力发展中的相互作用。 MyBP-C骨骼肌有三个 主要调节器：同工型（慢速与快速），剪接变体（长vs.短SMYBP-C）和翻译后 修饰（磷酸化）。 SMYBP-C在其N末端通过蛋白激酶A（PKA）磷酸化。这 SMYBP-C的作用，其在骨骼肌中的磷酸化和DA突变的作用尚不清楚。我们的初步 SMYBP-C的研究表明，与肌动球蛋白的结合取决于磷酸化和DA突变。我们 已经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， 肌肉球蛋白在肌肉中的相互作用以及磷酸化和突变的作用。我们的新初步研究 证明我们已经在N端子Smybp-C中成功开发了荧光传感器的结构 动力学对PKA介导的磷酸化敏感并与肌动蛋白结合。我们也发展了 分子间荧光测定法解决了FMYBP-C，长SMYBP-C和短暂的肌动蛋白结合 MYBP-C由于磷酸化和肌动蛋白上的肌球蛋白的存在。这些初步结果表明 不同骨骼MYBP-C调节的关键生理机制，并提供了其他科学 追求拟议研究的前提和可行性。 AIM 1将评估SMYBP-C结合的影响和 与肌动球蛋白相互作用，捕获N末端Smybp-C的结构和接近的DA突变， 肌动蛋白和肌球蛋白。光谱探针将被放置在这些蛋白质中，并将采用方法 在体外和原位检测野生型和DA突变体Smybp-C的钥匙构象。对于纤维实验， 肌肉将从新型SMYBP-C基因敲除（KO）小鼠中分离出来，并用重组SMYBP-透化 C，DA突变体和肌肉蛋白探针。样品将评估以结合和收缩功能。目的 2将确定PKA介导的SMYBP-C的磷酸化如何影响AIM 1中评估的参数。 AIM 3将确定FMYBP-C如何影响AIM 1中评估的参数，除了使用FMYBP-C KO和 用于纤维实验的SMYBP-C/FMYBP-C双KO小鼠。提出的研究捕获结构动力学 并使用新型的高分辨率方法实时和肌丝空间进行相互作用。这些目标 概述了在收缩周期中研究正常和突变骨骼MYBP-C的逐步计划。经过 监视蛋白质点的点与这些距离的顺序（或无序）之间的距离 生理条件，突变体可以分为垃圾箱，以促进靶向机械的疗法。