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.

肥厚性心肌病（HCM）患者患有高症状的伯恩，心力衰竭和 致命性心律不齐。虽然HCM被认为是肌节疾病已有30年了，但该疾病 肉瘤基因变异的机制尚未很好地定义，从而限制了治疗的精度和效率 选项。基因肌球蛋白结合蛋白C（MyBPC3）中的杂合变体导致所有情况中一半 家族性HCM。其中约15％是群集在内部蛋白质结构域C3和C6中的错义变体 具有不确定的绑定伙伴或功能的不确定。计算预测与我们的 已发布和初步的实验数据支持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 MYBP-C（由MyBPC3编码的蛋白质）的功能。在AIM 1中，我们将将涡轮接近标签应用于 野生型（WT）和突变体MyBP-C。在初步数据中，我们确定了> 200个新颖和独特的相邻 蛋白质至WT MYBP-C。将C3和C6突变体与野生型MYBP-C进行比较，相对丰度 肉瘤，细胞骨架和核糖核蛋白蛋白复合物减少，而核糖体和核糖体的丰度 伴侣蛋白增加。我们将通过评估这些改变的互动探索这些改变的后果 肌球蛋白构象，局部翻译和伴侣蛋白蛋白更新的变化。我们希望找到 存在与潜水功能的多种蛋白质的相互作用可以通过存在来加强或削弱 MYBP-C中的错义突变。克服与基因替代蛋白质相互作用的这种扰动 通过野生型MYBP-C是AIM 2的重点，我们将测试突变蛋白可以是的假设 野生型MYBP-C在肌膜中替换了石化。我们将翻译患者来源 表达具有腺相关病毒的C3或C6错义变体的诱导型心肌细胞 表达野生型MYBP-C或慢病毒矢量的向量表达“可滴定”野生型MYBP-C-C-FKBP12 融合蛋白可以实现剂量反应研究。结果度量将是突变体与 野生型蛋白质以及收缩和松弛速度。新的基因替代的体内研究 ARG506TRP MYBPC3敲入鼠标模型将完成HIPSC-CM实验。此应用程序 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 技术。这些目标的成功完成将通过定义扩展的MYBP-C中的新生物学 蛋白质邻域，通过揭示错义mybpc3变体的疾病机制，并通过测试 利用肌膜化学计量的内源性调节的基因位移策略，可能是 广泛适用于任何肌节基因中的错义变体。我们的调查团队由 高级，经验丰富的调查人员和有独特技能的初级调查员，被毒死了 实现这些目标。