Structural Dynamics of Cardiac Muscle Calcium ATPase Regulation

心肌钙 ATP 酶调节的结构动力学

• 批准号: 9981062
• 负责人: RAZVAN LIVIU CORNEA
• 金额: $ 78.23万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981062
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Academia; Affect; Alzheimer' s Disease; Amino Acid Sequence; Arrhythmia; Back; Biochemical; Biological Assay; Biology; Biophysical Process; Biophysics; Biosensor; Ca(2+)-Transporting ATPase; Calcium; Cardiac; Catalysis; Cells; Cellular Assay; Cellular biology; Chemicals; Chimeric Proteins; Clinical; Collaborations; Computer Simulation; Crystallography; Cysteine; Data; Detection; Diabetes Mellitus; Disease; Dwarfism; Engineering; Enzymes; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Future; Goals; Health; Heart; Heart Diseases; Heart failure; Human; Industry; Kinetics; Label; Lipids; Malignant Neoplasms; Mediating; Membrane; Membrane Proteins; Metabolic Diseases; Methods; Modeling; Molecular; Molecular Genetics; Motion; Motivation; Muscle; Muscle Cells; Muscular Dystrophies; Mutagenesis; Mutation; Myocardium; Nerve Degeneration; Obesity; Open Reading Frames; Optics; Parkinson Disease; Peptides; Phosphorylation; Physiological; Play; Protein Isoforms; Proteins; Public Health; Pump; Reader; Regulation; Research; Resolution; Roentgen Rays; Role; SERCA2a; Sarcoplasmic Reticulum; Scanning; Scientist; Site; Skeletal Muscle; Spectrum Analysis; Spin Labels; Structural Models; Structure; Techniques; Technology; Testing; Therapeutic; Time; Translating; Translational Research; Treatment Efficacy; Work; biophysical techniques; design; genetic regulatory protein; high throughput screening; innovation; insight; next generation; novel; phospholamban; protein structure; reconstitution; sarcolipin; sarcopenia; screening; small molecule; synergism; targeted treatment; therapeutic development; therapeutic target; tool

Project Summary/Abstract To create a roadmap for rational therapeutic design targeting Ca dysregulation, associated with heart failure and arrhythmia, we seek to understand the protein interactions and structural dynamics that regulate active Ca transport in cardiac muscle. Previous work focused on two membrane proteins, the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), in both skeletal and cardiac muscle, and its principal cardiac peptide subunit regulator: phospholamban (PLB). The project now focuses on the heart (SERCA2a isoform) and extends to additional peptide regulators. Our core technology is site-directed spectroscopy, in both purified proteins and living cells. We develop and apply innovative and complementary methods in site-directed labeling, fluorescence, EPR, and crystallography, with results integrated by computational simulations and function in biochemical and cellular assays. Aims 1&2 identify fundamental mechanisms, while Aim 3 combines these techniques and resultant insights to develop biophysical assays for therapeutic design. Aim 1 focuses on the functional dynamics of SERCA2a and its key structural transitions that mediate catalytic mechanism, focusing on steps that are critical for regulation in the heart. Added emphasis is now placed on detection of transient structural kinetics, using stopped-flow FRET methods pioneered by our lab, to directly relate SERCA structure and function. Aim 2 investigates mechanisms by which SERCA is regulated by three cardiac peptide subunits: PLB, sarcolipin (SLN), and DWORF. Aim 3 employs the insights of Aims 1&2 and our breakthroughs in high-throughput fluorescence detection, to implement novel small-molecule screening assays, with the ultimate goal of therapeutic discovery for heart disease. New compounds identified in Aim 3 feed back to provide mechanistic insight in Aims 1&2. Thus our Aims are synergistic, strengthening each other with new insights and hypotheses, yet not interdependent, since feasibility has been established independently for each Aim and sub-Aim. This project brings together a powerful and complementary combination of techniques and concepts, from biophysics to chemical biology to molecular genetics to cell biology, performed by a highly-integrated collaborative team, now adding a subcontract (Zima) to further enhance cellular and physiological relevance. The project remains grounded in fundamental biophysical mechanisms, and continues to exploit the recognized value of SERCA Ca pumps as therapeutic targets for major unmet needs in public health, targeting not only the heart, but also skeletal muscle (muscular dystrophy, sarcopenia), neurodegeneration (Alzheimer’s, Parkinson’s), and metabolic disease (diabetes, obesity). Thus, the significance of our work extends well beyond the heart. Our biophysical approaches will surely play a crucial role in understanding SERCA regulation, and also in controlling these functions. Our collaborators and consultants include scientists with expertise in therapeutic development, from academia and industry, and this project continues to stimulate separate efforts in truly translational research. 1

项目摘要/摘要 创建针对CA失调的理性热设计的路线图，与心力衰竭相关 和心律不齐，我们试图了解调节活性CA的蛋白质相互作用和结构动力学 心肌运输。以前的工作重点是两种膜蛋白，肌质网 （SR）CA-ATPase（SERCA），骨骼和心脏肌肉及其主要心脏肽亚基 调节剂：磷团（PLB）。该项目现在专注于心脏（SERCA2A同工型），并延伸到 其他肽调节剂。我们的核心技术是纯化的蛋白质的位置指导光谱 和活细胞。我们在站点定向的标签中开发并应用创新和完整的方法， 荧光，EPR和晶体学，其结果由计算模拟和功能集成 在生化和细胞测定中。目标1和2确定基本机制，而AIM 3结合了 这些技术和最终的见解，以开发用于治疗设计的生物物理测定法。目标1 重点关注SERCA2A的功能动力及其介导催化的关键结构过渡 机制，专注于对心脏调节至关重要的步骤。现在增加了重点 使用我们实验室开创的停止流fret方法检测瞬态结构动力学，直接 将SERCA结构和功能相关联。 AIM 2研究SERCA受三个调节的机制 心脏肽亚基：PLB，Sarcolipin（SLN）和Dworf。 AIM 3采用目标1和2的见解和 我们在高通量荧光检测中的突破，以实施新型的小分子筛选 测定法，是心脏病热发现的最终目标。 AIM 3中确定的新化合物 馈回目标1和2中提供机械见解。我们的目标是协同作用的，加强了每个 其他具有新的见解和假设，但没有相互依存，因为已经建立了可行性 独立于每个目标和sub-aim。该项目汇集了一个强大而完整的 从生物物理学到化学生物学再到分子遗传学再到细胞的技术和概念的结合 生物学，由一个高度综合的协作团队进行，现在添加分包合同（ZIMA）以进一步 增强细胞和身体相关性。该项目仍基于基本生物物理 机制，并继续探索SERCA CA泵作为治疗目标的公认价值 公共卫生的主要未满足需求，不仅针对心脏，而且针对骨骼肌肉（肌肉） 营养不良，肌肉减少症），神经变性（阿尔茨海默氏症，帕金森氏症）和代谢疾病（糖尿病，， 肥胖）。这，我们工作的意义远远超出了心脏。我们的生物物理方法 一定会在理解SERCA调节以及控制这些功能方面发挥至关重要的作用。 我们的合作者和顾问包括学术界具有专业知识的科学家 和行业，该项目继续在真正翻译的研究中刺激单独的努力。 1