STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN HEART

磷脂在心脏中的结构/功能

• 批准号: 6389254
• 负责人: LARRY R. JONES
• 金额: $ 38.65万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389254
• 关键词: active sites; affinity labeling; calcium transporting ATPase; cryoelectron microscopy; crystallization; dogs; electron spin resonance spectroscopy; enzyme inhibitors; enzyme reconstitution; fluorescence resonance energy transfer; genetically modified animals; heart contraction; isozymes; laboratory mouse; laboratory rabbit; liposomes; membrane proteins; phospholamban; phosphorylation; protein engineering; protein sequence; protein structure function; sarcoplasmic reticulum; site directed mutagenesis; tissue /cell culture

The long term goal of this research is to elucidate the mechanism by which phospholamban (PLB) regulates the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR which is composed of five identical monomers. Dephosphorylated PLB inhibits the Ca pump and Ca transport by SR, suppressing basal myocardial contractility. Phosphorylation of PLB during beta-adrenergic stimulation of the heart reverses Ca pump inhibition, augmenting contractility. For this renewal period, we will test the novel hypothesis that the PLB monomer is the active species inhibiting the Ca pump in the SR membrane. To test this hypothesis, four Specific Aims are proposed, which will examine PLB structure and function from the purified protein level to the level of the live animal. In Aim 1, the role of the PLB monomer in SERCA2a regulation will be investigated using co-expression of PLB with SERCA2a in Sf21 insect cells. The goal is to correlate the monomeric propensities of PLB mutants with the degree of SERCA2a inhibition. Taking advantage of this high-level expression system, we will resolve the kinetic step(s) regulated by PLB in the ATPase reaction scheme, and correlate changes in ATPase kinetics with PLB's effect on the rotational mobility of SERCA2a in the membrane. Aim 2 proposes to identify the sites of PLB:SERCA2a interaction in the membrane. This will be done by co- reconstituting SERCA2a into liposomes along with bioengineered PLB containing covalently attached photoaffinity- and spin-label probes. SERCA2a peptide fragments tagged with PLB photoaffinity probes will be sequenced. Residues of spin-labeled PLB in contact with the Ca pump will be identified by EPR spectroscopy. Aim 3 examines the dynamic equilibrium between PLB pentamers and monomers. The hypothesis will be tested that phosphorylation of PLB stabilizes monomeric mutants of PLB will be overexpressed in transgenic mouse hearts to assess the effects of the monomer on cardiac performance. Overexpression of superinhibitory PLB monomers in myocardium should yield dominant phenotypes exhibiting strong depression of contractility, conceivably leading to new animal models of heart failure. A total picture of PLB structure and function will emerge from the studies proposed. New insights on catecholamine regulation of the strength of the heartbeat will result.

这项研究的长期目标是通过 哪个磷脂（PLB）调节Ca泵的活性（SERCA2A 心脏肌浆网（SR）中的同工型）。 PLB是五个 心脏SR中的磷蛋白，由五个相同 单体。 去磷酸化的PLB抑制CA泵和CA转运 通过SR，抑制基底心肌收缩。 磷酸化 PLB在β-肾上腺素能刺激的心脏逆转CA泵 抑制，增加收缩力。 在这个续签时期，我们将 测试新的假设PLB单体是活性物种 抑制SR膜中的CA泵。 为了检验这一假设， 提出了四个具体目标，该目标将检查PLB结构和 从纯化的蛋白质水平到实时水平的功能 动物。 在AIM 1中，PLB单体在SERCA2A调节中的作用 将在SF21中使用PLB与SERCA2A共表达研究 昆虫细胞。 目的是将 具有SERCA2A抑制程度的PLB突变体。 利用 这个高级表达系统，我们将解决动力学步骤（S） 在ATPase反应方案中受PLB的调节，并将变化相关 在具有PLB对旋转迁移率的影响的ATPase动力学中 膜中的Serca2a。 AIM 2建议确定 PLB：膜中的SERCA2A相互作用。 这将由共同完成 将SERCA2A与生物工程PLB一起重新构成SERCA2A 包含共同附着的光性和自旋标签探针。 Serca2a肽片段用PLB光相处探针标记为 测序。 与CA泵接触的自旋标记PLB的残留物 将通过EPR光谱鉴定。 AIM 3检查动态 PLB五聚体和单体之间的平衡。 假设将是 测试了PLB的磷酸化稳定PLB的单体突变体 将在转基因小鼠心脏中过表达以评估效果 关于心脏性能的单体。 过表达 心肌中的超抑制性PLB单体应占主导地位 表现出强烈收缩性抑郁症的表型，可以想象 导致新的心力衰竭动物模型。 PLB的全部图片 结构和功能将从提出的研究中得出。 新的 关于心跳强度的儿茶酚胺调节的见解 会结果。