Calcium Signaling and Transport in S Cerevisiae

说明(申请人提供)：钙信号通路形成了一个网络，对人体和几乎所有真核生物中几乎所有细胞类型的正常运行都是至关重要的。阻断该网络不同组成部分的药物被用于治疗多种疾病。钙调神经磷酸酶是一种依赖于钙离子的蛋白磷酸酶，特异性阻断钙调神经磷酸酶的药物是有效的免疫抑制剂，广泛用于防止移植器官的免疫排斥反应。这项建议的主要目标是增加我们对钙调神经磷酸酶在钙信号网络中的调节和功能的理解。我们发现了一个新的蛋白质家族，它直接结合和调节钙调神经磷酸酶，从酵母到人类都是如此。通过对酵母的遗传学研究，我们提出了一种新的假说，即这些钙调神经磷酸酶(RCNs)在刺激和抑制状态之间循环，基于蛋白质中保守的连接器域的磷酸化。在这里，我们计划通过一系列仔细的生化和遗传学实验来批判性地评估这一假说，这些实验使用了我们最近发现的高度分化的RCN家族成员--人DSCR1/MCIP1、酵母Rcn1p和酵母Rcn2p。在酵母中，钙调神经磷酸酶抑制质膜高亲和力钙通道和液泡H+/钙离子交换，这有助于控制胞内钙离子和钙依赖酶如钙调神经磷酸酶的活性。我们已经确定了这些酶的几个新的亚基或调节因子，我们计划确定这些蛋白质之间的分子相互作用，哪些因子是钙调神经磷酸酶的直接底物，以及钙调神经磷酸酶的作用是否对这些酶的调节是必要的和充分的。最后，我们在酵母中发现了一个独特的低亲和力钙离子通道的第一个亚基或调节因子，并将其鉴定为人类claudin/stargazin膜蛋白超家族的成员，该家族已知在许多组织中调节离子通量。我们建议进行基因实验，以确定和表征酵母中这一新通道的其他成分和调节器。我们的发现将阐明真核生物钙信号网络的结构和功能，这可能会加速器官移植、心力衰竭治疗和耐药真菌感染治疗的改进疗法的发展。

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.