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 信号在横纹肌中达到最大强度和最高频率。维持它们的是钙结合蛋白 Casq，以其高钙结合能力、方便的亲和力和两种体外证明的独特特性：其结合 Ca 的能力明显依赖于游离 Ca 的含量除了这些 Ca 储存特性外，还存在 Ca 驱动的聚合倾向。 “门控”功能的证据，通过 Casq 感知周围的 Ca 浓度，并相应地通过机械连接诱导 Ca 释放通道 RyR 打开或关闭鉴于 Casq 中的多个突变，这些功能的相关性变得显而易见。我们的目标是定义这些与严重疾病相关的独特特性的体内运作。为此，我们加入了一个推动 Casq 物理化学研究的实验室。率先量化 Casq 在成人肌肉中的功能。该计划包括体外和体内测试。体内，三个假设：(1) 细胞中 Casq 聚合的程度和类型随着 [Ca] 耗尽而变化 (2) [Ca2+]SR 的变化通过 Casq 和 Tr 转化为门控变化。 RyR。 (3) Casq2 突变与 CPVT（儿茶酚胺多态性心室疾病）相关心动过速），以及 M87T，Casq1 的一种变体，在接受过心动过速测试的患者中存在相当大比例的患者。疾病MH（恶性高热），通过机制（1）和（2）引起疾病表型来测试。 (1) 我们将检查缺钙细胞中 Casq1 的 EM 结构，测试非聚合的能力突变的 Casq1 以恢复 Casq 缺失细胞的功能，并进行同位素 Ca 的体外测量扩散，探究 Casq 的存在是否会改变 Ca 扩散，以及这种影响如何取决于 Casq 对于（2），我们将探讨消除由提供的假定链接对 Ca 信号传导的影响。 (a) Tr 缺失小鼠和 (b) 细胞中的 Tr 通过表达“诱饵”（寡肽，对于 (3)，我们将表征 11 种已知的 Ca 依赖性物理化学。然后我们将测试 Casq2 的突变体、Casq1 的同源突变体及其 M87T 变体的能力。这些突变体恢复了 Casq 缺失小鼠纤维的功能，同时了解了这种标志性生物缓冲液的作用。作品将是主要奖励，长期目标是建立可转化为理性的基础科学基础解决人类疾病的策略，即：找到致病突变生成蛋白质→在体外表征其功能→然后在体内指定致病功能的获得或丧失。成功完成这些阶段将使我们能够在项目的未来迭代中设计可能救援策略测试其效果。