Calcium Signaling and Transport in S Cerevisiae

酿酒酵母中的钙信号传导和运输

• 批准号: 6988487
• 负责人: KYLE W CUNNINGHAM
• 金额: $ 34.87万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6988487
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; autoradiography; biological signal transduction; calcineurin; calcium channel; calcium flux; calcium transporting ATPase; cell membrane; enzyme activity; fungal genetics; fungal proteins; gene expression; mass spectrometry; membrane transport proteins; phosphorylation; protein kinase; protein protein interaction; protein structure function; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): Calcium signaling pathways form a network that is critical for normal operations of nearly all cell types in the human body and in nearly all eukaryotic species. Drugs that block different components of the network are used to therapeutically to treat many kinds of disorders. Drugs that specifically block calcineurin, a Ca2+- dependent protein phosphatase, are potent immunosuppressants used widely to prevent immunological rejection of transplanted organs. The major goal of this proposal is to increase our understanding of calcineurin regulation and functioning within the calcium signaling network. We discovered a novel family of proteins conserved from yeast to humans that directly bind and regulate calcineurin. Through genetic studies in yeast, we proposed a novel hypothesis where these regulators of calcineurin (RCNs) cycle between stimulatory and inhibitory states based on phosphorylation of a conserved linker domain in the proteins. Here we plan to critically evaluate this hypothesis in a careful series of biochemical and genetic experiments using human DSCR1/MCIP1, yeast Rcn1p, and yeast Rcn2p which we recently discovered as a highly divergent member of the RCN family. In yeast, calcineurin inhibits a plasma membrane high-affinity Ca2+ channel and a vacuolar H+/Ca2+ exchanger, which help to control cytosolic Ca2+ and the activities of Ca2+-dependent enzymes such as calcineurin. We have identified several new subunits or regulators of these enzymes and we plan to determine the molecular interactions among these proteins, which of the factors are direct substrates of calcineurin, and whether the effects of calcineurin are necessary and sufficient for regulation of the enzymes. Finally, we discovered the first subunit or regulator of a distinct low affinity Ca2+ channel in yeast and identified it as a member of the human claudin/stargazin superfamily of membrane proteins that are known to regulate ion fluxes in many tissues. We propose genetic experiments to identify and characterize additional components and regulators of this novel channel in yeast. Our findings will shed light on the structure and function of the calcium signaling network in eukaryotes, which may accelerate the development of improved therapeutics for organ transplantation, treatment of heart failure, and treatment of antibiotic-resistant fungal infections.

描述（由申请人提供）：钙信号通路形成一个网络，对于人体几乎所有细胞类型的正常操作至关重要，几乎所有真核物种中的正常操作至关重要。阻断网络不同组成部分的药物用于治疗多种疾病。特异性阻断钙调神经酶（一种Ca2+依赖性蛋白质磷酸酶）的药物是广泛用于防止移植器官的免疫抑制的有效免疫抑制剂。该提案的主要目标是增加我们对钙调节和钙信号网络中功能的理解。我们发现了一个新型的蛋白质家族，从酵母到人类保守，它们直接结合和调节钙调神经素。通过酵母中的遗传研究，我们提出了一个新的假设，基于蛋白质中保守的接头结构域的磷酸化，刺激性和抑制态之间的钙调神经酶（RCN）周期的调节剂。在这里，我们计划在使用人DSCR1/MCIP1，酵母RCN1P和酵母RCN2P的一系列仔细的生化和遗传实验中进行批判性评估这一假设。在酵母中，钙调蛋白抑制质膜高亲和力Ca2+通道和液泡H+/Ca2+交换剂，这有助于控制胞质CA2+以及Ca2+依赖性酶（如钙钙蛋白酶）的活性。我们已经确定了这些酶的几个新亚基或调节剂，并计划确定这些蛋白质之间的分子相互作用，这些因素是钙调神经素的直接底物，以及钙调蛋白的作用是否足以调节酶的调节。最后，我们在酵母中发现了独特的低亲和力Ca2+通道的第一个亚基或调节剂，并将其确定为膜蛋白的人类克劳丁/stargazin超家族的成员，这些膜蛋白已知可以调节许多组织中的离子通量。我们提出了遗传实验，以识别和表征酵母中这种新通道的其他成分和调节剂。我们的发现将阐明真核生物中钙信号网络的结构和功能，这可能会加速改善器官移植，心力衰竭以及治疗抗生素耐药真菌感染的治疗疗法。