Cardiac Calsequestrin Filament Dynamics in Catecholaminergic Polymorphic Ventricular Tachycardia

儿茶酚胺能多形性室性心动过速中的心脏 Calsequestrin 丝动力学

• 批准号: 9328651
• 负责人: Erron Wilcox Titus
• 金额: $ 3.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328651
• 关键词: Address; Affect; Age; Area; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Calcium; Calcium Binding; Calcium-Binding Proteins; Calsequestrin; Cardiac; Cardiac Myocytes; Catalogs; Catecholaminergic Polymorphic Ventricular Tachycardia; Cells; Classification; Crystallization; Dangerousness; Dimerization; Disease; Emotions; Exertion; Exhibits; Familial disease; Family; Family Study; Filament; Genes; Genetic; Genotype; Geometry; Heart; Heavy Metals; Heterozygote; Inheritance Patterns; Kinetics; Lead; Ligand Binding; Ligands; Maps; Metals; Missense Mutation; Modeling; Molecular Structure; Morbidity - disease rate; Mutation; Nature; Pathogenicity; Patients; Phenotype; Play; Protein Region; Proteins; Protomer; Publishing; Reporting; Resolution; Risk; Roentgen Rays; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Series; Site; Sodium Chloride; Strontium; Structure; Surface; System; Variant; Ventricular Arrhythmia; Water; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; aggressive therapy; base; biophysical analysis; biophysical properties; dimer; disease phenotype; experimental study; high risk; improved; induced pluripotent stem cell; innovation; molecular dynamics; mutant; novel; segregation; simulation; sudden cardiac death; variant of unknown significance

ABSTRACT Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial disorder in which polymorphic ventricular arrhythmias are caused by intensely-felt emotion or physical exertion. CPVT results from excess diastolic calcium leak and carries high risk of sudden cardiac death. On the basis of family studies, most cases of CPVT are associated with mutations in the cardiac ryanodine receptor (RYR2), the channel by which calcium exits the sarcoplasmic reticulum (SR), or cardiac calsequestrin (CASQ2), the principal calcium store of the SR. While RYR2-associated CPVT clearly exhibits autosomal dominant inheritance, the mode of inheritance of CASQ2-associated CPVT presents a puzzle. Many of the known CPVT-causing mutations appear to require recessive inheritance for penetrant disease, despite the fact that calsequestrin, a protein that must dimerize and then oligomerize to perform its function, would classically be considered vulnerable to functional disruption by heterozygous missense mutations. It is not known why some CASQ2 mutations, such as the recently reported K180R substitution, clearly have dominant deleterious effect, while others are pathogenic only when carried recessively. We propose to resolve this conundrum via a thorough structural and biophysical study of the cardiac calsequetrin filament. We have succeeded in determining what we believe is the first credible x-ray structure of the cardiac calsequestrin filament, revealing extensive buried surface area at oligomer contacts, as well as the filament's helical turn. We first hypothesize that the oligomer forms from a series of calcium salt bridges, such that mutations at calcium-binding sites make the oligomer particularly unstable. In our first aim, we propose to use x-ray crystallography with isomorphous calcium replacement to comprehensively map the calcium-binding sites of the calsequestrin filament, show that several known disease mutations are at ligand sites, and reveal that the oligomer requires calcium coordination in order to form. We also hypothesize that oligomer formation stabilizes dimers via extended all-by-all interactions, so that mutations that destabilize the calsequestrin dimer interface, when carried only in one copy, are rescued by the inherent stability of the oligomer at elevated SR-like calcium levels. In our second aim, we propose to demonstrate this rescue effect using biochemical assays and molecular dynamics simulations. In our third aim, we propose to model CPVT-causing CASQ2 mutations in iPS cells and demonstrate using a largely cell autonomous disease phenotype that the mutations that we have predicted on the basis of structure and dynamics to act in dominant fashion are in fact the ones that do so. The result of this project will be a new classification of CASQ2 mutations, an understanding of which types of mutations (oligomer interface vs dimer interface) have which disease inheritance mode and why, and a much improved basis for helping clinicians and patients decide whether aggressive therapy - which carries its own risks and morbidities - is warranted.

摘要 儿茶酚胺能多形性室性心动过速（CPVT）是一种家族性疾病， 室性心律失常是由强烈的情感或体力消耗引起的。CPVT由过量引起 心脏舒张期钙漏，导致心脏性猝死的风险很高。根据家庭研究，大多数案件 与心脏兰尼碱受体（RYR 2）的突变有关， 钙退出肌浆网（SR），或心脏钙螯合蛋白（CASQ 2），主要的钙储存， 虽然RYR 2相关的CPVT明显表现出常染色体显性遗传，但 CASQ 2相关的CPVT的遗传提出了一个难题。许多已知的引起CPVT的突变 似乎需要隐性遗传的渗透性疾病，尽管事实上，钙螯合蛋白， 必须二聚化，然后寡聚化以执行其功能，通常被认为易受 杂合错义突变导致的功能破坏。目前尚不清楚为什么一些CASQ 2突变，如 如最近报道的K180 R取代，明显具有主要的有害作用，而其他的则 只有在反复携带时才致病。我们建议通过彻底的结构和 心脏固钙素纤维的生物物理学研究。我们已经成功地确定了我们认为 第一个可靠的心脏钙螯合纤维的X射线结构，揭示了广泛的埋藏表面积， 低聚物接触，以及薄膜的螺旋转动。我们首先假设低聚物是由 一系列的钙盐桥，这样在钙结合位点的突变使寡聚体特别 不稳定在我们的第一个目标中，我们建议使用同晶钙替代的X射线晶体学， 全面绘制了钙螯合蛋白膜的钙结合位点，显示了几种已知的疾病 突变位于配体位点，并表明寡聚体需要钙配位才能形成。我们 我还假设寡聚体的形成通过扩展的所有相互作用稳定二聚体，因此， 当仅在一个拷贝中携带时，使钙螯合蛋白二聚体界面不稳定的突变被 低聚物在升高的SR样钙水平下的固有稳定性。在我们的第二个目标中，我们建议 使用生物化学测定和分子动力学模拟来证明这种拯救效果。在我们的第三 目的是，我们建议在iPS细胞中模拟CPVT引起的CASQ 2突变，并使用大量细胞 自主疾病表型，我们预测的突变的基础上的结构和 以主导方式行动的动力实际上是这样做的动力。该项目的结果将是一个新的 CASQ 2突变的分类，了解哪些类型的突变（寡聚体界面与二聚体） 接口）具有哪种疾病遗传模式和原因，以及帮助临床医生和 患者决定是否需要积极治疗--积极治疗本身也有风险和发病率。