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，对于人体和几乎所有真核物种中几乎所有细胞类型的正常运行至关重要。阻断网络不同组成部分的药物可用于治疗多种疾病。特异性阻断钙调神经磷酸酶（一种 Ca2+ 依赖性蛋白磷酸酶）的药物是有效的免疫抑制剂，广泛用于预防移植器官的免疫排斥。该提案的主要目标是增加我们对钙信号网络内钙调磷酸酶调节和功能的了解。我们发现了从酵母到人类都保守的一个新的蛋白质家族，它们直接结合和调节钙调神经磷酸酶。通过酵母的遗传学研究，我们提出了一个新的假设，即这些钙调磷酸酶 (RCN) 调节剂基于蛋白质中保守连接结构域的磷酸化在刺激状态和抑制状态之间循环。在这里，我们计划使用人类 DSCR1/MCIP1、酵母 Rcn1p 和酵母 Rcn2p（我们最近发现它们是 RCN 家族的高度分化成员）进行一系列仔细的生化和遗传实验，批判性地评估这一假设。在酵母中，钙调磷酸酶抑制质膜高亲和力 Ca2+ 通道和液泡 H+/Ca2+ 交换器，这有助于控制胞质 Ca2+ 和 Ca2+ 依赖性酶（如钙调磷酸酶）的活性。我们已经鉴定了这些酶的几个新亚基或调节因子，并计划确定这些蛋白质之间的分子相互作用，哪些因子是钙调磷酸酶的直接底物，以及钙调磷酸酶的作用对于酶的调节是否是必要和充分的。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器，并将其鉴定为人类膜蛋白 Claudin/stargazin 超家族的成员，已知该膜蛋白可调节许多组织中的离子通量。我们提出基因实验来鉴定和表征酵母中这种新通道的其他成分和调节剂。我们的研究结果将揭示真核生物中钙信号网络的结构和功能，这可能会加速器官移植、心力衰竭治疗和抗生素耐药真菌感染治疗的改进疗法的开发。