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以下方式阐明这一机制 哪种磷蛋白(PLB)调节钙泵(SERCA2a)的活性 在心肌肌浆网(SR)中。公共汽车是一种五聚体 心脏SR中的磷蛋白，由五个相同的 单体。去磷酸化的PLB抑制钙泵和钙转运 通过SR，抑制基础心肌收缩能力。磷酸化 β-肾上腺素能刺激心脏时的原球蛋白逆转钙泵 抑制，增强收缩能力。在此续期期间，我们将 检验PLB单体是活性物种的新假设 抑制SR膜上的钙泵。为了检验这一假设， 提出了四个具体目标，以审查公共汽车结构和 从纯化蛋白水平到活体水平的作用 动物。在目标1中，PLB单体在SERCA2a调节中的作用 将在Sf21中利用PLB和SERCA2a的共表达进行研究 昆虫细胞。我们的目标是将单体倾向与 PLb突变体具有SERCA2a抑制程度。利用 这个高水平的表达系统，我们将迈出解决的动力学步骤(S) 受PLB在ATPase反应过程中的调节，并关联变化 三磷酸腺苷酶动力学中原球蛋白对细胞旋转运动的影响 SERCA2a在细胞膜中。目标2建议确定 PLB：SERCA2a在膜上相互作用。这将由联合- SERCA2a与生物工程原球蛋白一起重组为脂质体 含有共价连接的光亲和自旋标记探针。 用PLB光亲和探针标记的SERCA2a多肽片段将被 已排序。自旋标记PLB与钙泵接触后的残留量 将通过电子顺磁共振波谱进行鉴定。《目标3》考察了 PLB五聚体和单体之间的平衡。假设将是 PLB的磷酸化稳定了PLB的单体突变体 将在转基因小鼠心脏中过表达以评估其效果 单体对心脏性能的影响。过度表达 心肌中的超抑制原球蛋白单体应占主导地位 可以想象，表现出强烈的收缩能力抑制的表型 导致了新的心力衰竭动物模型。公共小巴全景图 结构和功能将从拟议的研究中揭示出来。新的 儿茶酚胺调节心跳强度的研究 都会有结果。