Discovering the mechanisms and functions of signaling by the calcineurin beta1 isoform

项目摘要 全面绘制和监测信号通路对于实现 精准医学的目标。钙调神经磷酸酶(CN)、丝氨酸/苏氨酸蛋白磷酸酶和 免疫抑制剂的靶标FK506和CysA是钙依赖的关键介质 与人类健康相关的具有多种功能的信号。然而，许多CN监管的 底物和工艺仍有待阐明。这项建议的重点是CNb1，一个CN 具有独特性质和功能的异构体，在脊椎动物中和广泛保存 在人体组织中表达，但研究严重不足。通过解决以下方面的根本差距 了解CNb1，本研究将发现新的CN调控的信号通路， 阐明CNb1在健康和疾病细胞中的作用，并最终确定 以治疗性的方式操纵这种酶。我们的研究表明，它独特的C-尾巴是由 可选择的3‘端mRNA处理，赋予CNb1：in明显的调控和定位 在体外，在钙/钙调素存在的情况下，CNb1的最大磷酸酶活性显著 低于典型的CNb2，这是由于独特的C-末端自抑制序列 封闭一个重要的底物结合部位。在体内，CNb1定位于膜 与典型的CN异构体相比，包括质膜和高尔基体在内的隔室， 主要是胞质的。我们证明了保守的半胱氨酸在CNAb1C-Tail中的脂肪化 促进膜结合和CNAb1的棕榈酰化是动态的，提示 调节其在细胞中的分布和活性的新机制将在AIM中进行研究 1.CNb1不去磷酸化NFAT转录因子，其底物目前 未知。我们的中心假设是，通过绘制CNb1调节的信号通路，我们将 揭示CN在膜上的独特功能并提供对CN的关键新见解 在广泛的组织和过程中的监管。我们的初步数据表明 CNb1调节GPCR过程中质膜磷脂酰肌醇4-磷酸(PI4P)的合成 通过去磷酸化FAM126A发出信号，FAM126A的基因破坏导致 支持这一假说，这一假说进一步 在AIM 2上进行了测试。我们还提出了创新的方法，耦合TurboID以实现接近 利用CN结合肽的计算识别在快速时间帧上标记TO 系统地发现更多的CNb1底物，从而绘制这种酶的独特 AIM 3中的信令网络。我们预计这一知识将具有治疗应用 对于CN信号在其中起作用的病理，并将为其创建关键的新资源 研究钙离子或磷酸化依赖的信号传导的研究人员。

Project Summary Comprehensive mapping and monitoring of signaling pathways are essential for achieving the goals of precision medicine. Calcineurin (CN), the serine/threonine protein phosphatase and target of immunosuppressants, FK506 and CysA, is a critical mediator of Ca2+-dependent signaling with multiple functions relevant to human health. However, many CN-regulated substrates and processes remain to be elucidated. This proposal focuses on CNb1, a CN isoform with unique properties and functions that is conserved in vertebrates and broadly expressed in human tissues, but significantly under-studied. By addressing fundamental gaps in knowledge about CNb1, this research will discover novel CN-regulated signaling pathways, elucidate roles for CNb1 in healthy and diseased cells, and ultimately identify methods to therapeutically manipulate the enzyme. Our studies show that its unique C-tail, generated by alternative 3’ end mRNA processing, confers distinct regulation and localization to CNb1: In vitro, maximal phosphatase activity of CNb1 in the presence of Ca2+/calmodulin is significantly lower than that of canonical CNb2, due to a unique C-terminal auto-inhibitory sequence that occludes an essential substrate binding site. In vivo, CNb1 localizes to membrane compartments, including the plasma membrane and Golgi, in contrast to canonical CN isoforms, which are primarily cytosolic. We show that lipidation of conserved cysteines in the CNAb1 C-tail promotes membrane association and that palmitoylation of CNAb1 is dynamic, suggesting a novel mechanism for regulating its distribution and activity in cells that will be examined in Aim 1. CNb1 does not dephosphorylate NFAT transcription factors and its substrates are currently unknown. Our central hypothesis is that by mapping CNb1-regulated signaling pathways we will uncover unique functions for CN at membranes and provide critical new insights into CN regulation in a broad range of tissues and processes. Our preliminary data, which suggests that CNb1 regulates synthesis of phosphatidylinositol 4-phosphate (PI4P) at the PM during GPCR signaling by dephosphorylating FAM126A, whose genetic disruption gives rise to hypomyelination and congenital cataracts (HCC), supports this hypothesis, which is further tested in Aim 2. We also propose innovative approaches, coupling TurboID for proximity labeling over fast time frames with computational identification of CN-binding peptides, to systematically discover additional CNb1 substrates and thus map this enzymes’s unique signaling network in Aim 3. We anticipate that this knowledge will have therapeutic applications for pathologies to which CN signaling contributes, and will create a critical new resource for researchers studying Ca2+- or phosphorylation-dependent signaling.