Structural Determinants of Calcium Pump Regulation

钙泵调节的结构决定因素

基本信息

批准号：
7844209
负责人：
Seth L Robia
金额：
$ 26.55万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-15 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7844209
关键词：
Adrenergic Agents Affinity Binding Biology Ca(2+)-Transporting ATPase Cardiac Cause of Death Cells Complex Dependence Dilated Cardiomyopathy Disease Dissociation Epinephrine Equilibrium Figs - dietary Fluorescence Resonance Energy Transfer Fluorescent Probes Functional disorder Goals Heart Heart failure Human Kinetics Learning Left Life Link Measures Membrane Membrane Proteins Methods Modeling Molecular Molecular Conformation Mutagenesis Mutation Optical Methods Optics Phosphorylation Physiological Population Process Protein Binding Protein Biochemistry Protein Subunits Proteins Pump Recovery Regulation Relative (related person)Research Rest Reticulum Role Stimulus Structure System Techniques Testing Time United States adrenergic clinically significant gain of function inhibitor/antagonist interest loss of function monomer mutant novel phospholamban protein complex protein function protein protein interaction public health relevance research study response theories therapeutic target yellow-1

项目摘要

DESCRIPTION (provided by applicant): The applicant proposes a 2-AIM strategy to explore molecular mechanisms of SERCA calcium pump regulation by its major binding partner PLB. AIM1 describes the use of fluorescence resonance energy transfer (FRET) to quantify the AFFINITY of PLB binding interactions and the STRUCTURE of the resulting protein complexes. The proposed experiments directly measure the effect of phosphorylation and mutation of PLB on its regulatory interactions. Because the fluorescent probes are genetically encoded, observations of these membrane protein interactions are being made in living cells for the first time. AIM2 proposes to measure the PROTEIN BINDING KINETICS of the membrane protein complexes important for cardiac Ca2+ regulation. The rate of formation and dissolution of these membrane protein complexes is an important determinant of their function, but these rates are inaccessible to classical protein-protein binding kinetics methods. The applicant has invented a new optical method that can measure membrane protein subunit exchange dynamics in living cells. PUBLIC HEALTH RELEVANCE: The proteins PLB and SERCA are of clinical significance because of their central role in cardiac function and disease. Disorders of these proteins are associated with heart failure, and loss or mutation of PLB in humans results in the disease "dilated cardiomyopathy". Thus, PLB is a regarded as a high value therapeutic target in the treatment of heart failure, a leading cause of death in the United States.

描述（由申请人提供）：申请人提出了一种2-AIM策略来探索SERCA钙泵通过其主要结合伴侣PLB调节的分子机制。 AIM1 描述了使用荧光共振能量转移 (FRET) 来量化 PLB 结合相互作用的亲和力以及所得蛋白质复合物的结构。所提出的实验直接测量 PLB 的磷酸化和突变对其调节相互作用的影响。由于荧光探针是基因编码的，因此首次在活细胞中观察到这些膜蛋白相互作用。 AIM2 建议测量对心脏 Ca2+ 调节很重要的膜蛋白复合物的蛋白结合动力学。这些膜蛋白复合物的形成和溶解速率是其功能的重要决定因素，但经典的蛋白质-蛋白质结合动力学方法无法获得这些速率。申请人发明了一种新的光学方法，可以测量活细胞中膜蛋白亚基交换动态。公众健康相关性：PLB 和 SERCA 蛋白具有临床意义，因为它们在心脏功能和疾病中发挥着核心作用。这些蛋白质的紊乱与心力衰竭有关，人类 PLB 的缺失或突变会导致“扩张型心肌病”。因此，PLB 被认为是治疗心力衰竭的高价值治疗靶点，而心力衰竭是美国的主要死亡原因。