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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

基本信息

批准号：
9922363
负责人：
Erron Wilcox Titus
金额：
$ 0.42万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9922363
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 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

项目摘要

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.

抽象的儿茶酚胺能多形性室性心动过速 (CPVT) 是一种家族性疾病，其中多形性室性心动过速室性心律失常是由强烈的情绪或体力消耗引起的。 CPVT 源于过量舒张期钙渗漏并具有心源性猝死的高风险。大多数情况下，在家庭研究的基础上 CPVT 的发生与心脏兰尼碱受体 (RYR2) 的突变有关，该通道通过钙离开肌浆网 (SR) 或心脏钙钙蛋白 (CASQ2)，这是钙的主要储存 SR。虽然 RYR2 相关的 CPVT 明显表现出常染色体显性遗传，但 CASQ2 相关 CPVT 的遗传带来了一个难题。许多已知的 CPVT 引起突变渗透性疾病似乎需要隐性遗传，尽管事实上钙螯合蛋白（一种蛋白质）必须先二聚然后寡聚才能发挥其功能，通常被认为容易受到杂合错义突变造成的功能破坏。目前尚不清楚为什么某些 CASQ2 突变，例如如最近报道的 K180R 替代品，显然具有显着的有害作用，而其他则仅当隐性携带时才致病。我们建议通过彻底的结构性和心脏钙叶蛋白丝的生物物理学研究。我们已经成功地确定了我们所相信的是什么心脏钙铁丝的第一个可靠的 X 射线结构，揭示了广泛的埋藏表面区域低聚物接触，以及细丝的螺旋转弯。我们首先假设低聚物是由一系列钙盐桥，使得钙结合位点的突变使寡聚物特别不稳定。在我们的第一个目标中，我们建议使用 X 射线晶体学和同晶钙替代来全面绘制了 calsequestrin 丝的钙结合位点图，表明几种已知的疾病突变发生在配体位点，表明寡聚物需要钙配位才能形成。我们还假设寡聚体的形成通过扩展的所有相互作用来稳定二聚体，因此当仅在一个副本中携带时，破坏钙螯合蛋白二聚体界面稳定性的突变可通过低聚物在升高的 SR 样钙水平下具有固有的稳定性。在我们的第二个目标中，我们建议使用生化测定和分子动力学模拟证明了这种救援效果。在我们的第三个目标，我们建议对 iPS 细胞中引起 CPVT 的 CASQ2 突变进行建模，并使用大量细胞进行演示自主疾病表型，即我们根据结构和预测的突变事实上，以主导方式行事的动力才是这样做的动力。该项目的成果将是一个新的 CASQ2 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）有哪些疾病遗传方式及其原因，为帮助临床医生和患者提供了很大的基础。患者决定是否需要采取积极的治疗——这种治疗有其自身的风险和发病率。