Structural Dynamics of Cardiac Muscle Calcium ATPase Regulation

项目摘要/摘要 为针对与心力衰竭相关的钙调节失调的合理治疗设计创建路线图 和心律失常，我们试图了解调节活动钙离子的蛋白质相互作用和结构动力学 在心肌中运输。以前的工作主要集中在肌浆网这两种膜蛋白上。 骨骼肌和心肌中的(SR)Ca-ATPase(SERCA)及其主要的心脏多肽亚基 调节剂：磷脂蛋白(PLB)。该项目现在专注于心脏(SERCA2a亚型)，并扩展到 额外的多肽调节剂。我们的核心技术是定点光谱学，在这两种纯化的蛋白质中 和活细胞。我们开发和应用创新和互补的方法在现场定向标记， 荧光、EPR和结晶学，结果通过计算模拟和函数进行整合 在生化和细胞分析中。目标1和目标2确定基本机制，而目标3结合 这些技术和由此产生的见解，以开发用于治疗设计的生物物理分析。目标1 重点研究SERCA2a的功能动力学及其介导催化的关键结构转变 机制，重点放在心脏调节的关键步骤上。现在更强调的是 使用我们实验室首创的停流FRET方法检测瞬时结构动力学，以直接 联系SERCA的结构和功能。目的2研究SERCA受三种基因调控的机制 心脏多肽亚基：PLB、肌磷脂(SLN)和DWORF。目标3借鉴了目标1和2的见解，并 我们在高通量荧光检测方面的突破，实现了新型的小分子筛选 检测，最终目标是发现心脏病的治疗方法。AIM 3中鉴定的新化合物 反馈，在目标1和目标2中提供机械性的洞察力。因此，我们的目标是协同的，相互加强 其他有新的见解和假设，但不相互依赖，因为可行性已经确立 独立于每个目标和子目标。这个项目汇集了一个强大和互补的 技术和概念的结合，从生物物理到化学生物学，从分子遗传学到细胞 生物，由一个高度集成的协作团队执行，现在增加了一个分包合同(ZIMA)以进一步 增强细胞和生理相关性。该项目仍然以基础生物物理学为基础。 机制，并继续利用SERCA钙泵作为治疗靶点的公认价值 公共卫生中未得到满足的主要需求，不仅针对心脏，而且针对骨骼肌(肌肉 营养不良、骨质疏松症)、神经变性(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 将在理解SERCA调控和控制这些功能方面发挥关键作用。 我们的合作者和顾问包括在治疗开发方面具有专业知识的科学家，来自学术界 和行业，这个项目继续刺激在真正的翻译研究方面的单独努力。 1

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