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

发现钙调神经磷酸酶 beta1 亚型的信号传导机制和功能

• 批准号: 9921418
• 负责人: Martha S. Cyert
• 金额: $ 39.15万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9921418
• 关键词: 1-Phosphatidylinositol 4-Kinase; Address; Adverse effects; Binding; Binding Sites; Biochemical; Biotin; C-terminal; Calcineurin; Calcineurin inhibitor; Calmodulin; Catalytic Domain; Cell membrane; Cells; Communities; Complex; Coupling; Cysteine; Data; Defect; Diabetes Mellitus; Disease; Distributional Activity; Enzymes; Etiology; FK506; G Protein-Coupled Receptor Signaling; Genetic; Goals; Golgi Apparatus; Health; Heart Diseases; Human; Immune System Diseases; Immunosuppressive Agents; In Vitro; Intracellular Membranes; Isoenzymes; Knowledge; Label; Letters; Ligase; Lipids; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Messenger RNA; Methods; Monitor; Organ Transplantation; PPP3CA gene; PPP3CB gene; Pathology; Peptides; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Process; Production; Property; Protein Isoforms; Protein phosphatase; Proteins; Regulation; Research; Research Personnel; Resources; Role; Schizophrenia; Serine; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Tail; Testing; Therapeutic; Threonine; Time; Tissues; Transplant Recipients; Vertebrates; Work; congenital cataract; experience; human tissue; in vivo; innovation; insight; novel; novel therapeutic intervention; palmitoylation; phosphatidylinositol 4-phosphate; precision medicine; research study; transcription factor

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.

项目摘要 信号通路的全面测绘和监测对于实现 精准医疗的目标钙调神经磷酸酶（CN），丝氨酸/苏氨酸蛋白磷酸酶和 免疫抑制剂的靶点FK 506和CysA是Ca 2+依赖性免疫应答的关键介质， 具有与人类健康相关的多种功能的信号传导。然而，许多受氯化萘管制的 基质和方法仍有待阐明。该提案侧重于CNb 1，即CN 具有独特性质和功能的同种型，在脊椎动物中保守， 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， 关于CNb 1的知识，这项研究将发现新的CN调节信号通路， 阐明CNb 1在健康和患病细胞中的作用，并最终确定方法， 治疗性地操纵酶。我们的研究表明，其独特的C-尾，产生的 替代3'末端mRNA加工，赋予CNb 1不同的调节和定位： 在体外，CNb 1在Ca 2 +/钙调素存在下的最大磷酸酶活性显著高于在Ca 2 +/钙调素存在下的最大磷酸酶活性。 低于标准CNb 2，这是由于独特的C-末端自抑制序列， 封闭了必需的底物结合位点。在体内，CNb 1定位于膜 隔室，包括质膜和高尔基体，与典型的CN亚型相反， 主要是胞质的。我们发现CNAb 1 C-尾中保守半胱氨酸的脂化 促进膜结合，CNAb 1的棕榈酰化是动态的，这表明 调节其在细胞中分布和活性的新机制，将在Aim中进行研究 1. CNb 1不能使NFAT转录因子去磷酸化，其底物目前是 未知我们的中心假设是，通过绘制CNb 1调节的信号通路， 揭示CN在膜上的独特功能，并提供对CN的重要新见解 调节广泛的组织和过程。我们的初步数据表明， CNb 1调节GPCR期间PM处磷脂酰肌醇4-磷酸（PI 4P）的合成 通过去磷酸化FAM 126 A信号传导，其遗传破坏引起 髓鞘形成不足和先天性白内障（HCC），支持这一假设，这是进一步 目标2测试我们还提出了创新的方法，耦合TurboID的接近 通过计算识别CN结合肽在快速时间范围内进行标记， 系统地发现额外的CNb 1底物，从而绘制这种酶的独特的 Aim 3中的信令网络。我们预计，这一知识将有治疗应用 对于CN信号导致的病理学，并将为 研究Ca 2+或磷酸化依赖信号的研究人员。