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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.

项目总结