Structural Dynamics of Cardiac Muscle Calcium ATPase Regulation

心肌钙 ATP 酶调节的结构动力学

• 批准号: 10474966
• 负责人: RAZVAN LIVIU CORNEA
• 金额: $ 77.31万
• 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/10474966
• 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; 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; therapeutically effective; 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

项目概要/摘要 制定针对与心力衰竭相关的 Ca 失调的合理治疗设计路线图 和心律失常，我们寻求了解调节活性 Ca 的蛋白质相互作用和结构动力学 在心肌中的运输。之前的工作重点关注两种膜蛋白，即肌浆网 (SR) Ca-ATP 酶 (SERCA)，存在于骨骼肌和心肌中，及其主要心脏肽亚基 调节剂：受磷蛋白（PLB）。该项目现在重点关注心脏（SERCA2a 同工型）并扩展到 额外的肽调节剂。我们的核心技术是定点光谱学，适用于纯化的蛋白质 和活细胞。我们在定点标签中开发并应用创新和补充方法， 荧光、EPR 和晶体学，通过计算模拟和函数整合结果 在生化和细胞测定中。目标 1 和 2 确定了基本机制，而目标 3 则结合了 这些技术和由此产生的见解可以开发用于治疗设计的生物物理测定。目标1 重点关注 SERCA2a 的功能动力学及其介导催化作用的关键结构转变 机制，重点关注对心脏调节至关重要的步骤。现在更加强调 使用我们实验室首创的停流 FRET 方法检测瞬态结构动力学，直接 关联 SERCA 结构和功能。目标 2 研究 SERCA 受三个因素调节的机制 心脏肽亚基：PLB、肌磷脂 (SLN) 和 DWORF。目标 3 采用了目标 1 和 2 的见解， 我们在高通量荧光检测方面取得突破，实现新型小分子筛选 检测，最终目标是发现心脏病的治疗方法。目标 3 中发现的新化合物 反馈以提供目标 1 和 2 中的机制见解。因此，我们的目标是协同的，加强每个目标 其他具有新的见解和假设，但并不相互依赖，因为可行性已经确立 每个目标和子目标都是独立的。该项目汇集了强大且互补的 技术和概念的结合，从生物物理学到化学生物学到分子遗传学到细胞 生物学，由高度集成的协作团队执行，现在添加分包合同（Zima）以进一步 增强细胞和生理相关性。该项目仍然以基本的生物物理学为基础 机制，并继续利用 SERCA Ca 泵作为治疗靶点的公认价值 公共卫生方面未满足的主要需求，不仅针对心脏，还针对骨骼肌（肌肉 营养不良、肌肉减少症）、神经退行性疾病（阿尔茨海默氏症、帕金森氏症）和代谢性疾病（糖尿病、 肥胖）。因此，我们工作的意义远远超出了内心。我们的生物物理方法 肯定会在理解 SERCA 调控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来自学术界的具有治疗开发专业知识的科学家 和工业界，这个项目继续刺激真正的转化研究的单独努力。 1