Structural Determinants of Calcium Pump Regulation

钙泵调节的结构决定因素

基本信息

批准号：
8103106
负责人：
Seth L Robia
金额：
$ 33.41万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8103106
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 Fluorescence Resonance Energy Transfer Fluorescent Probes Functional disorder Goals Health 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 research study response theories therapeutic target

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，以探索其主要结合伴侣PLB的Serca钙泵调节的分子机制。 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和所得蛋白质复合物的结构。提出的实验直接测量了PLB对其调节相互作用的磷酸化和突变的影响。由于荧光探针是遗传编码的，因此首次在活细胞中对这些膜蛋白相互作用进行观察。 AIM2建议测量膜蛋白复合物的蛋白结合动力学对于心脏CA2+调节很重要。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，但是对于经典蛋白质 - 蛋白质结合动力学方法而言，这些速率无法接近。申请人发明了一种新的光学方法，该方法可以测量活细胞中的膜蛋白亚基交换动力学。公共卫生相关性：蛋白质PLB和SERCA具有临床意义，因为它们在心脏功能和疾病中的核心作用。这些蛋白质的疾病与心力衰竭有关，人类PLB的丧失或突变导致疾病“扩张的心肌病”。因此，PLB在治疗心力衰竭的治疗方面被认为是高价值的治疗靶标，这是美国的主要死亡原因。