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

基本信息

批准号：
10818737
负责人：
Martha S. Cyert
金额：
$ 8.89万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10818737
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 Explosion 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 Nuclear Pore Complex Peptides Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Process Protein Dephosphorylation Protein Isoforms Protein Kinase Protein phosphatase Proteins Regulation Research Role Saccharomyces cerevisiae Seizures Signal Pathway Signal Transduction Tissues Work adaptive immune response 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

项目摘要

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.

项目摘要所有真核生物均使用基于磷酸化的信号网络，由蛋白质激酶和磷酸酶，以调节细胞过程。而有关激酶信号的全球信息具有爆炸后，对磷酸酶的了解落后了。缺乏系统的，公正的方法为了分析磷酸酶信号传导在我们对细胞调节的理解中留下了重大差距网络。在> 25年以上，我对钙调神经酶的研究，保守的Ca2+/钙调蛋白调节酿酒酵母和人类中的磷酸酶直接解决了这个问题。钙调神经酶无处不在在心血管，神经和免疫系统中表达并扮演关键的调节作用（1）；但是，目前仅建立了约70种蛋白质作为钙调蛋白底物（2）。钙调神经素去磷酸化激活的T细胞（NFAT）转录因子的核因子激活自适应免疫反应（3）和钙调神经蛋白抑制剂FK506和环孢菌素A通常是规定为免疫抑制剂（4）。但是，这些药物会引起不良影响，包括高血压，糖尿病和癫痫发作是通过抑制非免疫组织中的钙调神经蛋白（5）的（5）需要系统地绘制人类钙调神经蛋白信号通路。我的工作阐明了钙调神经素通过发现短线性图案（Slims）的新方法传导：短在内在疾病区域内发现的肽序列，这些序列确定了特定的，低 - 信号传导必不可少的亲和力相互作用（6）。使用实验和基于硅细的方法，我们最近定义了人类钙调蛋白信号网络（2）。这项工作发现了通过钙调神经酶对核孔复合物的保守调节，并显示出意外的钙调蛋白靠近中心体（2）。未来的努力将在这些方面阐明Ca2+和钙调神经酶信号传导使用包括磷光蛋白质组学和接近标记的方法组合的隔室带有更快的标记生物素连接酶（Miniturbo（33），分裂涡轮增压）。荧光传感器将用于在体内这些位置的探针Ca2+信号。膜的钙调神经酶功能将通过专注于CNB1，这是一种有点研究的同工型，其独特的，独特的本地化和调节脂化的C末端序列（10）。通过动态棕榈酰化来调节CNB1活性检查，CNB1特异性信号通路（包括PI4P合成调节）将是旨在实现对该酶的全面和机械理解。最后，新技术，Mrble-Pep（微球比率条形码灯笼编码编码为耦合到肽）（11），将开发用于定量分析和纤细的发现。总体而言，这项研究旨在系统地绘制人类钙调神经蛋白信号通路，并提高我们对基于细长的信号传导更广泛。