Redox signaling in axon guidance: Structure and activity of MICAL

轴突引导中的氧化还原信号传导：MICAL 的结构和活性

• 批准号: 7862630
• 负责人: L. Mario Amzel
• 金额: $ 40.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862630
• 关键词: Actins; Affinity; Agreement; Axon; Binding; Biological; Biological Process; Brain; C-terminal; C2 Domain; Cardiac; Cell Adhesion; Cell Culture Techniques; Cell membrane; Cells; Cellular Morphology; Chemicals; Chemotaxis; Co-Immunoprecipitations; Complex; Cues; Cultured Cells; Cytoskeletal Modeling; Cytoskeleton; Data; Destinations; Development; Differential Scanning Calorimetry; Down-Regulation; Drosophila genus; Electrons; Epigallocatechin Gallate; Equilibrium; Family; Goals; Growth; Growth Cones; Homologous Gene; Hydrogen Peroxide; Immune system; In Vitro; Individual; Injury; Integrins; Intervention; LIM Domain; Length; Ligand Binding; Measures; Mediating; Mediator of activation protein; Methods; Mixed Function Oxygenases; Modification; Molecular; Molecular Conformation; Mus; Muscle fasciculation; N-terminal; NADP; Nature; Nerve Regeneration; Neuraxis; Neuroglia; Neurons; Neuropilins; Oxidation-Reduction; Oxygen; Play; Process; Production; Proline-Rich Domain; Proteins; Reaction; Reactive Oxygen Species; Reducing Agents; Regulation; Reporting; Research; Resolution; Rest; Role; SH3 Domains; Semaphorin-3A; Semaphorins; Shapes; Signal Transduction; Skeletal Development; Source; Structure; Techniques; Testing; Therapeutic Intervention; Titrations; Transfection; Ultracentrifugation; Work; angiogenesis; axon guidance; base; calponin; cancer cell; cell motility; design; flavin-containing monooxygenase; inhibitor/antagonist; intermolecular interaction; mutant; nerve injury; neurodevelopment; plexin; public health relevance; receptor; research study; response; stoichiometry; three dimensional structure

During neural development, axons are guided to their final destination by a large number of molecular cues-attractive and repulsive signals that instruct the cytoskeleton to redirect the direction of growth. One of these signals involves the interaction of Semaphorins with Plexin. The presence of this signal is conveyed to the cytoskeleton by another molecule, MICAL (Molecule Interacting with CasL), a multidomain protein with an FAD-containing hydroxylase (MICALfd ) domain. We have determined the structure of MICALfd and showed that it catalyzes the reduction of O2 to H20 2 using NADPH. This activity is inhibited by EGCG, a monooxygenase inhibitor that also inhibits the repulsive action of Semaphorins. It has been shown that H20 2 production by MICAL in cell culture correlates with cell contraction, an activity associated with cytoskeletal reorganization. It was shown that the H20 2 production is a highly regulated process involving interaction of the FAD domain with other MICAL domains and controlled by interaction with the C2 domain of Plexin and with CRMP (collapsin response mediator protein). In addition, the identified interaction of MICAL with CasL has been shown to be an essential component of the regulation of defasciculation, a key early process in axon guidance. The long term goal of this project is to use biophysical methods and atomic resolution 3D structure determination to characterize the regulation of MICAL activity. In this project we concentrate on the interactions among MICAL domains and between MICAL and CasL. Our aims are: 1) to uncover the mechanism by which intramolecular interactions autoinhibit H20 2 production and regulate MICAL redox activity, and 2) to identify and characterize the interactions of MICAL with the SH3 domain of CasL that control defasciculation. Axon guidance signals playa major role in the development of the central nervous system and in nerve regeneration after injury. A detailed characterization of the interactions responsible for these signals is essential for understanding brain development and for the design of pharmacological interventions after nerve injuries.

在神经发育过程中，轴突被大量分子线索引导至最终目的地——吸引和排斥信号，指示细胞骨架重新调整生长方向。这些信号之一涉及信号蛋白与 Plexin 的相互作用。该信号的存在通过另一种分子 MICAL（与 Ca​​sL 相互作用的分子）传递到细胞骨架，MICAL 是一种具有包含 FAD 的羟化酶 (MICALfd) 结构域的多结构域蛋白。我们已经确定了 MICALfd 的结构，并表明它利用 NADPH 催化 O2 还原为 H20 2 。这种活性受到 EGCG 的抑制，EGCG 是一种单加氧酶抑制剂，也能抑制信号蛋白的排斥作用。研究表明，细胞培养物中 MICAL 产生的 H20 2 与细胞收缩相关，细胞收缩是与细胞骨架重组相关的活动。结果表明，H20 2 的产生是一个高度调控的过程，涉及 FAD 结构域与其他 MICAL 结构域的相互作用，并由与 Plexin 的 C2 结构域和 CRMP（崩溃蛋白反应介导蛋白）的相互作用控制。此外，已发现的 MICAL 与 CasL 的相互作用已被证明是解束调节的重要组成部分，解束是轴突引导的关键早期过程。该项目的长期目标是利用生物物理方法和原子分辨率3D结构测定来表征MICAL活性的调节。在这个项目中，我们专注于 MICAL 域之间以及 MICAL 和 CasL 之间的相互作用。我们的目标是：1) 揭示分子内相互作用自动抑制 H20 2 产生并调节 MICAL 氧化还原活性的机制，2) 识别和表征 MICAL 与控制解束的 CasL SH3 结构域的相互作用。 轴突引导信号在中枢神经系统的发育和损伤后的神经再生中发挥着重要作用。详细表征这些信号的相互作用对于理解大脑发育和神经损伤后药物干预的设计至关重要。