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) 患者会经历高症状负担、心力衰竭和 致死性心律失常 虽然 HCM 被认为是一种肌节疾病已超过 30 年，但该疾病 肌节基因变异的机制尚未明确，限制了治疗的精度和功效 肌球蛋白结合蛋白 C (MYBPC3) 基因的杂合变异导致一半病例。 其中大约 15% 是聚集在内部蛋白结构域 C3 和 C6 的错义变异。 与我们结合的计算预测具有不确定的结合伙伴或功能。 已发表的和初步的实验数据支持以下假设：C3 和 C6 结构域导致多种蛋白质-蛋白质相互作用的扰动，这对于正常的蛋白质相互作用至关重要。 MyBP-C（由 MYBPC3 编码的蛋白质）的功能 在目标 1 中，我们将应用 TurboID 邻近标记。 在初步数据中，我们已经鉴定出超过 200 个新颖且独特的邻近蛋白。 与 WT MyBP-C 相比，C3 和 C6 突变体与野生型 MyBP-C 的相对丰度。 肌节、细胞骨架和核糖核蛋白复合物减少，而核糖体和核糖核蛋白复合物的丰度降低 我们将通过评估来探索这些相互作用的后果。 我们期望发现肌球蛋白构象、局部翻译和伴侣介导的蛋白质周转的变化。 与具有不同功能的多种蛋白质的相互作用会因存在而增强或减弱 MyBP-C 中的错义突变克服了蛋白质相互作用与基因替换的干扰。 野生型 MyBP-C 是目标 2 的重点，我们将测试突变蛋白可以是 我们将在肌节内用野生型 MyBP-C 进行化学计量替换。 与腺相关病毒一起表达 C3 或 C6 错义变体的诱导型多能心肌细胞 表达野生型MyBP-C的载体或表达“可滴定”野生型MyBP-C-FKBP12的慢病毒载体 能够进行剂量反应研究的融合蛋白，结果测量将是突变体与突变体的摩尔比。 野生型蛋白质以及新的基因替换的体内研究。 Arg506Trp MYBPC3 敲入小鼠模型将补充 hiPSC-CM 实验这一应用。 探索 MyBP-C 生物学的几个新颖方面，并具有独特的试剂和先进的蛋白质组学 成功完成这些目标将通过定义扩展来揭示 MyBP-C 中的新生物学。 蛋白质邻域，通过揭示错义 MYBPC3 变异的疾病机制，并通过测试 利用肌节化学计量的内源性调节的基因置换策略，可以是 广泛适用于任何肌节基因中的错义变异。我们的研究团队由多种人员组成。 经验丰富的资深调查员和具有独特技能的才华横溢的初级调查员，已做好准备 实现这些目标。