Calcium Signaling and Transport in S Cerevisiae

描述（由申请人提供）：钙信号通路形成一个网络，该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作至关重要。阻断网络不同组成部分的药物被用于治疗多种疾病。特异性阻断钙调磷酸酶（一种钙依赖性蛋白磷酸酶）的药物是广泛用于预防移植器官免疫排斥反应的强效免疫抑制剂。这项提案的主要目标是增加我们对钙信号网络中钙调神经磷酸酶调节和功能的理解。我们发现了一个新的蛋白质家族，从酵母到人类，直接结合和调节钙调神经磷酸酶。通过在酵母中的遗传学研究，我们提出了一种新的假设，这些调节剂的钙调神经磷酸酶（RCN）之间的刺激和抑制状态的基础上磷酸化的保守连接结构域的蛋白质。在这里，我们计划严格评估这一假设，在一系列仔细的生化和遗传实验中，使用人类DSCR 1/MCIP 1，酵母Rcn 1 p和酵母Rcn 2 p，我们最近发现的RCN家族的一个高度分歧的成员。在酵母中，钙调神经磷酸酶抑制质膜高亲和力Ca 2+通道和液泡H+/Ca 2+交换器，这有助于控制胞质Ca 2+和Ca 2+依赖性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剂，我们计划确定这些蛋白质之间的分子相互作用，哪些因素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是必要的和足够的酶的调节。最后，我们发现了酵母中独特的低亲和力Ca 2+通道的第一个亚基或调节剂，并将其鉴定为已知在许多组织中调节离子通量的人封闭蛋白/stargazin膜蛋白超家族的成员。我们提出了遗传实验，以确定和表征额外的组件和监管机构，这种新的渠道在酵母。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，心力衰竭治疗和耐药性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.