Missense Variants in Myosin Binding Protein C that Cause Hypertrophic Cardiomyopathy

导致肥厚性心肌病的肌球蛋白结合蛋白 C 的错义变异

• 批准号: 10752380
• 负责人: Sharlene M Day
• 金额: $ 71.68万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752380
• 关键词: Affect; Alleles; Arrhythmia; Binding; Binding Proteins; Biological Assay; Biological Process; Biology; Cardiac Myocytes; Chimeric Proteins; Clinical Trials; Complement; Complex; Congestive Heart Failure; Cytoskeleton; DNA Sequence Alteration; Data; Disease; Disease Pathway; Dose; Echocardiography; Electrostatics; Eligibility Determination; Familial Hypertrophic Cardiomyopathy; Gene Delivery; Genes; Genetic; Goals; Heart; Heart failure; Heterozygote; Human; Hypertrophic Cardiomyopathy; Knock-in Mouse; Label; Laboratories; Lead; Lentivirus Vector; Mass Spectrum Analysis; Measures; Mediating; Missense Mutation; Molecular Chaperones; Molecular Conformation; Mutate; Mutation; Myocardium; Myofibrils; Myosin ATPase; Neighborhoods; Outcome Measure; Pathogenicity; Patients; Proteins; Proteomics; Publishing; Reagent; Regulation; Relaxation; Research Personnel; Ribonucleoproteins; Ribosomes; Sarcomeres; Surface Properties; Tacrolimus Binding Protein 1A; Talents; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Translations; Treatment Efficacy; Variant; Viral Vector; adeno-associated viral vector; delivery vehicle; experience; experimental study; gene replacement; gene replacement therapy; gene therapy; genetic variant; improved; in vivo; induced pluripotent stem cell; insight; loss of function; mouse model; mutant; myosin-binding protein C; novel; prevent; primary outcome; protein degradation; protein protein interaction; response; secondary outcome; skills; stoichiometry; success; sudden cardiac death

Patients with hypertrophic cardiomyopathy (HCM) experience a high symptomatic burden, heart failure and lethal arrhythmias. While HCM has been recognized as a disease of the sarcomere for >30 years, the disease mechanisms for sarcomeric gene variants are not well defined, limiting the precision and efficacy of treatment options. Heterozygous variants in the gene myosin-binding protein C (MYBPC3) cause half of all cases of familial HCM. About 15% of these are missense variants that cluster in interior protein domains C3 and C6 which have uncertain binding partners or function. Computational predictions combined with our published and preliminary experimental data support the hypothesis that missense variants in C3 and C6 domains lead to perturbation of multiple protein-protein interactions that are critical for the normal function of MyBP-C (the protein encoded by MYBPC3). In Aim 1 we will apply TurboID proximity labeling to wild-type (WT) and mutant MyBP-C. In preliminary data we have identified >200 novel and unique neighboring proteins to WT MyBP-C. Comparing C3 and C6 mutants to wild-type MyBP-C, relative abundances of sarcomeric, cytoskeletal and ribonucleoprotein complexes are reduced, while abundances of ribosomal and chaperone proteins are increased. We will explore consequences of these altered interactions by assessing changes in myosin conformation, local translation, and chaperone-mediate protein turnover. We expect to find that interactions with multiple proteins of diverse function are either strengthened or weakened by the presence of missense mutations in MyBP-C. Overcoming this perturbation in protein interactions with gene replacement by wild-type MyBP-C is the focus of Aim 2 where we will test the hypothesis that the mutant protein can be stoichiometrically replaced within the sarcomere by wild-type MyBP-C. We will transduce patient-derived inducible-pluripotent cardiomyocytes expressing C3 or C6 missense variants with adeno-associated viral vectors expressing wild-type MyBP-C or a lentiviral vector expressing a “titratable” wild-type MyBP-C-FKBP12 fusion protein that enables dose-response studies. The outcome measures will be the molar ratio of mutant to wild-type protein, and contractile and relaxation velocities. In vivo studies of gene replacement in a new Arg506Trp MYBPC3 knock-in mouse model will complement the hiPSC-CM experiments. This application explores several novel aspects of MyBP-C biology and features unique reagents and advanced proteomic techniques. Successful completion of these aims will uncover new biology in MyBP-C by defining an expanded protein neighborhood, by revealing disease mechanisms for missense MYBPC3 variants, and by testing a gene displacement strategy that leverages endogenous regulation of sarcomeric stoichiometry and could be broadly applicable to missense variants in any sarcomere gene. Our investigative team, composed of a mix of senior, highly experienced investigators and talented junior investigators with unique skill sets, is well poised to achieve these goals.

肥厚型心肌病（HCM）患者的症状负担高，心力衰竭， 致命的心律失常虽然HCM被认为是肌节疾病已有>30年，但该疾病 肌节基因变异的机制尚未明确，限制了治疗的精确性和有效性 选项.肌球蛋白结合蛋白C（MYBPC 3）基因的杂合变体导致所有病例的一半。 家族性HCM其中约15%是错义变体，聚集在内部蛋白质结构域C3和C6中 其具有不确定的结合伴侣或功能。计算预测结合我们的 已发表的和初步的实验数据支持这一假设，即在C3和 C6结构域导致多种蛋白质-蛋白质相互作用的扰动，这些相互作用对正常的 MyBP-C（由MYBPC 3编码的蛋白质）的功能。在目标1中，我们将应用TurboID邻近标记来 野生型（WT）和突变型MyBP-C。在初步数据中，我们已经确定了>200个新颖和独特的邻居 WT MyBP-C。比较C3和C6突变体与野生型MyBP-C， 肌节、细胞骨架和核糖核蛋白复合物减少，而核糖体和 伴侣蛋白增加。我们将通过评估这些改变的相互作用的后果， 肌球蛋白构象、局部翻译和分子伴侣介导的蛋白质周转的变化。我们希望能找到 不同功能的多种蛋白质之间的相互作用， MyBP-C中的错义突变。通过基因替换克服蛋白质相互作用中的这种扰动 野生型MyBP-C是目标2的重点，我们将测试突变蛋白可以被 在肌节内被野生型MyBP-C化学计量取代。我们将以患者为导向， 表达C3或C6错义变体的可诱导多能心肌细胞与腺相关病毒 表达野生型MyBP-C的载体或表达“可滴定的”野生型MyBP-C-FKBP 12的慢病毒载体 融合蛋白，使剂量反应研究。结果测量将是突变体与蛋白质的摩尔比。 野生型蛋白质，以及收缩和舒张速度。新基因替代的体内研究 Arg 506 Trp MYBPC 3基因敲入小鼠模型将补充hiPSC-CM实验。本申请 探索MyBP-C生物学的几个新方面，并提供独特的试剂和先进的蛋白质组学 技术.这些目标的成功完成将通过定义一个扩展的 通过揭示错义MYBPC 3变异体的疾病机制，并通过测试MYBPC 3基因的突变， 利用内源性调节肌节化学计量的基因置换策略， 广泛适用于任何肌节基因中的错义变体。我们的调查小组由 资深、经验丰富的调查员和具有独特技能的有才华的初级调查员， 以实现这些目标。