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)患者经历高症状负担、心力衰竭和 致命的心律失常。虽然肥厚型心肌炎被认为是一种肌节疾病已有30年的历史，但这种疾病 肉瘤基因变异的机制尚未明确，限制了治疗的精确度和有效性。 选择。肌球蛋白结合蛋白C(MYBPC3)基因的杂合变异导致一半的病例 家族性肉芽肿病。其中约15%是错义变体，聚集在内部蛋白质结构域C3和C6中 它们具有不确定的结合伙伴或功能。计算预测与我们的 已发表的和初步的实验数据支持这一假设，即C3和C3中的错义变体 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(MYBPC3编码的蛋白)的功能。在目标1中，我们将应用TurboID邻近标记来 野生型(WT)和突变体MyBP-C。在初步数据中，我们已经确定了&gt；200新的和独特的邻居 WT MyBP-C的蛋白质。比较C3和C6突变体与野生型MyBP-C，相对丰度 肌节、细胞骨架和核糖核蛋白复合体减少，而核糖体和 伴侣蛋白增加。我们将通过评估这些变化的交互作用来探索其后果 肌球蛋白构象、局部翻译和伴侣介导的蛋白质周转的变化。我们希望能找到 与多种功能不同的蛋白质的相互作用因存在而增强或减弱 MyBP-C的错义突变。用基因替换克服蛋白质相互作用中的这种扰动 通过野生型MyBP-C是目标2的焦点，在那里我们将检验突变蛋白可以 肌节内的化学计量比由野生型MyBP-C取代。我们将转导患者衍生的 用腺相关病毒诱导表达C3或C6错义变异体的多能心肌细胞 表达野生型MyBP-C的载体或表达可滴定的野生型MyBP-C-FKBP12的慢病毒载体 能够进行剂量反应研究的融合蛋白。结果衡量标准将是突变株与 野生型蛋白质，以及收缩和松弛速度。一种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。此应用程序 探索MyBP-C生物学的几个新方面，并以独特的试剂和高级蛋白质组为特色 技巧。成功完成这些目标将在MyBP-C中发现新的生物学，通过定义扩展的 通过揭示MYBPC3错义变体的致病机制，以及通过测试一种 利用肌节化学计量的内源性调节的基因置换策略，并可能是 广泛适用于任何肌节基因的错义变体。我们的调查小组，由一群 经验丰富的高级调查员和具有独特技能的才华横溢的初级调查员，准备好 实现这些目标。