Roles of Calsequestrin in the Control of Calcium Signals in Health and Disease

Calsequestrin 在控制健康和疾病钙信号中的作用

基本信息

批准号：
8928639
负责人：
CHULHEE KANG
金额：
$ 34.37万
依托单位：
RUSH UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8928639
关键词：
Address Adult Affinity Anabolism Basic Science Binding Buffers Calcium Calcium Binding Calcium Signaling Calsequestrin Canada Cardiac Cell physiology Cells Clinical Complex Computer Simulation Dependence Deposition Diagnosis Diffusion Disease Failure Fiber Foundations Frequencies Functional disorder Future Goals Health Heart Diseases Hepatocyte Homologous Gene Human Image In Vitro Individual Ions Knockout Mice Lead Life Link Malignant hyperpyrexia due to anesthesia Measurement Measures Mechanics Mediating Modification Molecular Movement Mus Muscle Mutate Mutation Myocardium Neurons Null Lymphocytes Oligopeptides Outcome Patients Permeability Phenotype Physical Chemistry Polymers Property Protein Isoforms Proteins Proteolysis Public Health Recombinants Resources Retrieval Rewards Role Signal Transduction Signaling Molecule Skeletal Muscle Solutions Specific qualifier value Staging Stretching Striated Muscles Structure Structure-Activity Relationship Techniques Testing Tissues Translating Triad Acrylic Resin Variant Ventricular Tachycardia Work design disease phenotype functional gain functional loss functional restoration human disease human subject in vitro testing in vivo mouse junctate protein mutant operation polymerization prevent research study skeletal skills stem triadin vector

项目摘要

Intracellular Ca signals reach their greatest intensity and highest frequencies in striated muscle. Sustaining them is calsequestrin, Casq, notable for its high Ca-binding capacity, convenient affinity and for two unique properties demonstrated in vitro: a marked dependence of its ability to bind Ca on how much free Ca is present and a Ca-driven tendency to polymerize. In addition to these Ca storing properties, there are evidences for a “gating” function, whereby Casq senses the surrounding Ca concentration and accordingly induces the Ca release channel, RyR, to open or close, through a mechanical link presumably provided by triadin, Tr. The relevance of these functions becomes obvious in view of multiple mutations in Casq that are linked to grave diseases. Our goal is to define the operation in vivo of these unique properties demonstrated in vitro. To this end we joined a lab that has driven the study of Casq's physical chemistry and one that pioneered the quantification of Casq's functions in adult muscle. The plan includes the testing, in vitro and in vivo, of three hypotheses: (1) the extent and type of polymerization of Casq in cells changes as [Ca] depletes inside the cellular store (SR). (2) Changes in [Ca2+]SR are translated, via Casq and Tr, to gating changes in the RyR. (3) Mutations in Casq2 linked to the disease CPVT (catecholaminergic polymorphic ventricular tachycardia), as well as M87T, a variant in Casq1 present in a sizable percentage of patients tested for the disease MH (malignant hyperthermia), cause the disease phenotype through mechanisms (1) and (2). To test (1) we will examine the EM structure of Casq1 in Ca-depleted cells, test the ability of a non-polymerizing mutated Casq1 to restore function in Casq-null cells, and carry out in vitro measurements of isotopic Ca diffusion, probing whether the presence of Casq alters Ca diffusion, and how the effects depends on Casq polymerization. For (2) we will explore the effects on Ca signaling of eliminating the putative link provided by Tr in (a) Tr-null mice and (b) cells acutely deprived of the link by expression of “decoys”, the oligopeptides that bind Casq in the Tr sequence. For (3) we will characterize the Ca-dependent physical chemistry of 11 known mutants of Casq2, the homologous mutants of Casq1 and its M87T variant. We will then test the ability of these mutants to restore function in Casq-null mouse fibers. While understanding how this iconic biobuffer works will be a main reward, the long-term goal is to build a basic science foundation translatable to rational strategies that address human diseases, namely: find the causative mutationgenerate the proteincharacterize its function in vitro  then in vivo specify the pathogenic functional gain or loss. Successful completion of these stages will allow us in future iterations of the project to design possible rescue strategiestest their efficacy.

细胞内CA信号达到了最大的强度和最高的频率。维持它们的是CalSequestin，Casq，其高CA结合能力，方便的亲和力以及两个在体外证明的独特特性：其绑定CA的能力的明显依赖性是自由ca的数量当前和CA驱动的聚合趋势。除了这些CA存储属性外，还有 “门控”功能的证据，从而使CASQ感觉到周围的CA浓度并因此诱导CA释放通道RYR，通过大概由 Triadin，tr。鉴于CASQ中的多个突变是，这些功能的相关性变得显而易见与严重疾病有关。我们的目标是在体内定义这些独特特性的操作体外。为此，我们加入了一个实验室，该实验室推动了Casq的物理化学反应的研究开创了CASQ在成人肌肉中功能的定量。该计划包括测试，体外和体内三个假设的体内：（1）CASQ在细胞中的聚合的程度和类型随着[CA]的耗竭而变化在蜂窝存储（SR）内。（2）[Ca2+] SR的变化通过CASQ和TR转换为门控变化里尔。（3）与疾病CPVT相关的CASQ2突变（儿茶酚胺能多态性心室心动过速）以及M87T，这是CASQ1中的一种变体，其中很大一部分的患者被测试疾病MH（恶性高温），通过机制（1）和（2）导致疾病表型。测试（1）我们将检查CA缺失细胞中CASQ1的EM结构，测试非聚合化的能力突变的CASQ1恢复CASQ-NULL细胞中的功能，并对同位素CA进行体外测量扩散，探测CASQ的存在是否改变了Ca扩散以及效果如何依赖CASQ 聚合。对于（2），我们将探讨消除CA信号的影响在（a）Tr-null小鼠和（b）细胞中，TR被“诱饵”的表达急剧剥夺了连接，这是寡肽在TR序列中结合CASQ。对于（3），我们将表征11个已知的CA依赖性物理化学性质 Casq2的突变体，Casq1的同源突变体及其M87T变体。然后，我们将测试这些突变体可以恢复CASQ-NULL小鼠纤维中的功能。同时了解这种标志性的生物固定器作品将是主要的奖励，长期目标是建立一个可以翻译成理性的基础科学基金会解决人类疾病的策略，即：找到原因突变 蛋白质将其功能在体外然后体内指定致病功能增益或损失。这些阶段的成功完成将使我们在将来的项目迭代中进行设计可能救援策略测试其效率。