Systematic elucidation of calcineurin phosphatase signaling in humans

系统阐明人类钙调神经磷酸酶信号传导

• 批准号: 10371219
• 负责人: Martha S. Cyert
• 金额: $ 54.91万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10371219
• 关键词: Address; Affinity; Bar Codes; Biochemical; Biotin; C-terminal; Calcineurin; Calcineurin inhibitor; Calmodulin; Cardiovascular system; Cell physiology; Cells; Centrosome; Coupled; Cyclosporine; Diabetes Mellitus; Disease; Enzymes; Epilepsy; Eukaryota; FK506; Future; Heart Diseases; Human; Hypertension; Immune System Diseases; Immune system; Immunosuppressive Agents; Knowledge; Label; Ligase; Location; Malignant Neoplasms; Maps; Mediating; Membrane; Methods; Microspheres; Nervous system structure; Nuclear Pore Complex; Peptides; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Process; Protein Isoforms; Protein Kinase; Protein phosphatase; Proteins; Regulation; Research; Role; Saccharomyces cerevisiae; Seizures; Signal Pathway; Signal Transduction; Tissues; Work; adaptive immune response; base; calcineurin phosphatase; in silico; in vivo; insight; new technology; novel strategies; novel therapeutic intervention; nuclear factors of activated T-cells; palmitoylation; phosphoproteomics; protein aminoacid sequence; ratiometric; research study; sensor; transcription factor

Project Summary All eukaryotes use phosphorylation-based signaling networks, composed of protein kinases and phosphatases, to regulate cellular processes. While global information about kinase signaling has exploded, knowledge of phosphatases has lagged behind. The lack of systematic, unbiased approaches to analyze phosphatase signaling leaves a major gap in our understanding of cellular regulatory networks. For >25 years, my research on calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, in S. cerevisiae and humans has directly addressed this issue. Calcineurin is ubiquitously expressed and plays critical regulatory roles in the cardiovascular, nervous and immune systems (1); however, only ~70 proteins are currently established as calcineurin substrates (2). Calcineurin dephosphorylates the Nuclear Factor of Activated T-cells (NFAT) transcription factors to activate the adaptive immune response (3), and calcineurin inhibitors, FK506 and cyclosporin A, are commonly prescribed as immunosuppressants (4). However, these drugs cause unwanted effects, including hypertension, diabetes, and seizures by inhibiting calcineurin in non-immune tissues (5), highlighting the need to map human calcineurin signaling pathways systematically. My work elucidates calcineurin signaling through novel approaches based on the discovery of short linear motifs (SLiMs): short degenerate peptide sequences found within regions of intrinsic disorder that determine specific, low- affinity interactions that are essential for signaling (6). Using experimental and in silico SLiM-based methods, we recently defined the human calcineurin signaling network (2). This work uncovered conserved regulation of nuclear pore complexes by calcineurin and showed unexpected calcineurin proximity to centrosomes (2). Future efforts will elucidate Ca2+ and calcineurin signaling at these compartments using a combination of methods that include phosphoproteomics and proximity labeling with faster-labeling biotin ligases (miniTurbo (33), Split TurboID). Fluorescent sensors will be used to probe Ca2+ signals at these locations in vivo. Calcineurin functions at membranes will be analyzed by focusing on CNb1, a little-studied isoform with distinct localization and regulation conferred by its unique, lipidated C-terminal sequences (10). Regulation of CNb1 activity via dynamic palmitoylation will be examined, and CNb1-specific signaling pathways, including regulation of PI4P synthesis, will be established to achieve a comprehensive and mechanistic understanding of this enzyme. Finally, the novel technology, MRBLE-PEP (Microspheres Ratiometric Barcode Lanthenide Encoding coupled to PEPtides) (11), will be developed for quantitative analysis and discovery of SLiMs. Overall this research aims to map human calcineurin signaling pathways systematically and to advance our understanding of SLiM-based signaling more broadly.

项目概要 所有真核生物都使用基于磷酸化的信号网络，由蛋白激酶和 磷酸酶，调节细胞过程。虽然有关激酶信号传导的全球信息已 随着爆炸式增长，磷酸酶的知识已经落后。缺乏系统的、公正的方法 分析磷酸酶信号传导在我们对细胞调节的理解上留下了重大空白 网络。超过 25 年以来，我对钙调神经磷酸酶（一种保守的 Ca2+/钙调蛋白调节蛋白）的研究 酿酒酵母和人类中的磷酸酶直接解决了这个问题。钙调磷酸酶无处不在 在心血管、神经和免疫系统中表达并发挥关键的调节作用 (1)； 然而，目前仅约 70 种蛋白质被确定为钙调神经磷酸酶底物 (2)。钙调磷酸酶 使活化 T 细胞核因子 (NFAT) 转录因子去磷酸化，以激活 适应性免疫反应 (3) 以及钙调神经磷酸酶抑制剂 FK506 和环孢菌素 A 是常见的药物 规定为免疫抑制剂（4）。然而，这些药物会引起不良反应，包括 通过抑制非免疫组织中的钙调磷酸酶来预防高血压、糖尿病和癫痫发作 (5)，强调 需要系统地绘制人类钙调磷酸酶信号通路。我的工作阐明了钙调神经磷酸酶 通过基于短线性基序 (SLiMs) 发现的新方法进行信号传导：短 在内在紊乱区域内发现的简并肽序列决定了特异性、低 对于信号传导至关重要的亲和力相互作用 (6)。使用基于 SLiM 的实验和计算机模拟 方法，我们最近定义了人类钙调神经磷酸酶信号网络 (2)。这部作品揭开了 钙调磷酸酶对核孔复合物的保守调节并显示出意想不到的钙调磷酸酶 接近中心体 (2)。未来的努力将阐明这些方面的 Ca2+ 和钙调神经磷酸酶信号传导 使用磷酸蛋白质组学和邻近标记等方法组合进行隔室 使用标记速度更快的生物素连接酶（miniTurbo (33)、Split TurboID）。荧光传感器将用于 探测体内这些位置的 Ca2+ 信号。钙调神经磷酸酶在膜上的功能将通过以下方式进行分析 重点关注 CNb1，这是一种很少被研究的异构体，具有独特的定位和调节作用， 脂化 C 末端序列 (10)。通过动态棕榈酰化调节 CNb1 活性将是 检查，CNb1 特异性信号通路，包括 PI4P 合成的调节，将被 建立的目的是为了对这种酶有全面和机械的了解。最后， 新技术，MRBLE-PEP（微球比率条码镧系元素编码耦合到 PEPtides) (11)，将用于 SLiM 的定量分析和发现。总体而言，本研究 旨在系统地绘制人类钙调神经磷酸酶信号通路并增进我们对 更广泛的基于 SLiM 的信号传输。