STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN HEART

磷脂在心脏中的结构/功能

• 批准号: 2695294
• 负责人: LARRY R. JONES
• 金额: $ 23.17万
• 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/2695294
• 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阐明机制， 受磷蛋白（PLB）调节钙泵（SERCA 2a）的活性 同种型）。PLB是五聚体 心脏SR中的磷蛋白由五个相同的 单体。 去磷酸化PLB抑制钙泵和钙转运 通过SR，抑制基础心肌收缩力。 磷酸化 β-肾上腺素能刺激心脏时PLB逆转Ca泵 抑制增强收缩力 在此更新期间，我们将 测试PLB单体是活性物质的新假设 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，这些目标将研究公共小巴的结构， 从纯化蛋白水平到肝脏水平的功能 动物 在目的1中，PLB单体在SERCA 2a调节中的作用 将使用PLB与SERCA 2a在Sf 21中的共表达来研究 昆虫细胞 目标是将聚合物的单体倾向 PLB突变体具有SERCA 2a抑制程度。 利用 这个高水平的表达系统，我们将解决动力学步骤（S） 在ATP酶反应图式中受PLB调节， 在ATP酶动力学中，PLB对 膜中的SERCA 2a。 目标2建议确定 PLB：膜中的SERCA 2a相互作用。 这将由共同完成- 与生物工程PLB一起将SERCA 2a重构为脂质体沿着 含有共价连接的光亲和和自旋标记探针。 用PLB光亲和探针标记的SERCA 2a肽片段将被标记。 测序 与Ca泵接触的自旋标记PLB的残留物 将通过EPR光谱进行鉴定。目标3检查动态 PLB五聚体和单体之间的平衡。 假设是 测试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度表达，以评估 对心脏功能的影响。 过表达 心肌中的超抑制性PLB单体应产生占主导地位的 表现出强烈的收缩性抑制的表型， 导致新的心力衰竭动物模型。 公共小巴全貌 结构和功能将从拟议的研究中得出。 新 关于儿茶酚胺调节心跳强度的见解 将导致。