RyR architecture and gatekeeping of internal calcium stores

RyR 结构和内部钙存储的把关

• 批准号: 10252844
• 负责人: Montserrat Samso
• 金额: $ 37.89万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10252844
• 关键词: 3-Dimensional; Address; Adopted; Affect; Anesthetics; Architecture; Arrhythmia; Buffers; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cell Line; Cells; Central Core Myopathy; Cessation of life; Classification; Complex; Coupling; Cryoelectron Microscopy; Cytoplasmic Structures; Cytoplasmic Tail; Dantrolene; Data Set; Defect; Development; Dihydropyridine Receptors; Disease; Distant; Drug Design; Electron Microscope; Electron Microscopy; Electrons; Etiology; Exertion; Future; Gatekeeping; Generations; Glutathione Disulfide; Heart; Heart Diseases; Heart failure; Heterogeneity; Homeostasis; Hydration status; Ions; Knowledge; Lead; Length; Life; Link; Malignant hyperpyrexia due to anesthesia; Measurable; Mediating; Mitochondria; Modification; Molecular; Molecular Conformation; Multiminicore disease; Muscle Fatigue; Musculoskeletal; Mutation; Myocardium; Myopathy; Nemaline Myopathies; New Agents; Oryctolagus cuniculus; Outcome; Oxidative Stress; Oxides; Pathology; Pharmaceutical Preparations; Physiological; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Relaxation; Resolution; Rest; Rhabdomyolysis; Role; RyR1; RyR2; Ryanodine Receptor Calcium Release Channel; Sampling; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; Sodium Chloride; Statistical Data Interpretation; Structure; Syndrome; Technology; Testing; Three-Dimensional Image; Time; Tissues; Transducers; Transmembrane Domain; Vestibule; alpha helix; base; design; detector; image reconstruction; molecular rearrangement; muscle aging; mutant; oxidation; particle; premature; release of sequestered calcium ion into cytoplasm; skeletal; structural biology; three dimensional structure

PROJECT SUMMARY The intracellular, large conductance intracellular RyR calcium release channel is a major regulator of calcium homeostasis in skeletal and cardiac muscle cells. Naturally occurring mutations in the skeletal isoform RyR1 lead to several myopathies (central core disease, multi-minicore disease, nemaline myopathy, exertional rhabdomyolysis) and malignant hyperthermia, whereas mutations in the cardiac isoform RyR2 lead to arrhythmia and heart failure. Some post-translational modifications can also cause similar effects in wild type RyR1 and RyR2. The vast majority of these RyR alterations cause disruption of calcium homeostasis through RyR hypersensitization, which causes sub- threshold opening and sarcoplasmic reticulum calcium leak. The project applies state-of-the-art structural biology technology to determine RyR's structure and allosterism at the atomic level and in a near-native state, in order to examine the molecular mechanism of RyR-mediated calcium leak and analyze how similar modifications affect differently the two RyR isoforms. We will determine the atomic structure of selected RyR1 and RyR2 mutants, and wt RyR1 or RyR2 with post-translational modifications, using cryoEM and 3D image reconstruction. Reaching atomic resolution of non-crystalline samples by cryoEM is now possible owing to the recent development of direct electron detectors for electron microscopy. To further establish and characterize possible heterogeneity in the dataset, classification and multivariate statistical analysis of several thousand RyR cryoEM particles will be performed. Mutant RyRs will be purified from HEK cell lines, and post-translational modifications will be studied on RyR purified from rabbit. Solving the atomic structure of RyR with disease-related alterations will help to understand the molecular mechanism of defective channel closure, and reveal any differential mechanisms between the skeletal and cardiac isoforms. This will advance the field forward towards structure-based, isoform-specific drug design.

项目摘要 细胞内的大电导细胞内RyR钙释放通道是一个主要的 骨骼肌和心肌细胞中钙稳态的调节剂。天然存在的突变 在骨骼同种型中，RyR1导致几种肌病（中央核心病，多微核心病， 线状体肌病、劳力性横纹肌溶解症）和恶性高热，而 心脏同种型RyR2导致心律失常和心力衰竭。一些翻译后修饰 也可以在野生型RyR1和RyR2中引起类似的效应。绝大多数RyR变异 通过RyR超敏作用引起钙稳态的破坏，从而引起亚 阈值开放和肌浆网钙渗漏。 该项目应用最先进的结构生物学技术来确定RyR的结构， 在原子水平和近天然状态下的变构，以检查分子 RyR介导的钙渗漏的机制，并分析类似的修饰如何不同地影响 两种RyR亚型。我们将确定选定的RyR1和RyR2突变体的原子结构， 具有翻译后修饰的野生型RyR1或RyR2，使用cryoEM和3D图像重建。 通过cryoEM达到非晶体样品的原子分辨率现在是可能的，这是由于最近的 电子显微镜直接电子探测器的发展。进一步建立 描述数据集、分类和多元统计分析中可能的异质性， 将进行数千次RyR cryoEM颗粒。将从HEK细胞中纯化突变RyR 系，并将对从兔中纯化的RyR进行翻译后修饰研究。 解决RyR的原子结构与疾病相关的改变将有助于理解 有缺陷的通道关闭的分子机制，并揭示任何差异机制之间 骨骼和心脏的同种型这将推动该领域朝着基于结构， 异构体特异性药物设计。

项目成果

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

• DOI: 10.1128/mbio.02270-22
• 发表时间: 2022-12-20
• 期刊: mBio
• 影响因子: 6.4

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells

HEK293 细胞中表达的重组野生型和突变型兰尼碱受体的纯化

• DOI: 10.21769/bioprotoc.4112
• 发表时间: 2021
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Hu Yifan;Iyer Kavita;Nayak Ashok;Kurebayashi Nagomi;Murayama Takashi;Samso Montserrat
• 通讯作者: Samso Montserrat

Cryo-EM catalyzes the exploration of drug selectivity: The CDK7 inhibitor example.

冷冻电镜催化药物选择性的探索：CDK7 抑制剂示例。

• DOI: 10.1016/j.bpj.2021.02.010
• 发表时间: 2021
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Samsó,Montserrat