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: 7661822
• 负责人: L. Mario Amzel
• 金额: $ 38.37万
• 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/7661822
• 关键词: Affinity; Axon; Back; Binding; Biological; Biological Process; Blood; Brain; C-terminal; C2 Domain; Cardiac; Cell Adhesion; Cell Culture Techniques; Cell membrane; Cells; Cellular Morphology; Cellular biology; Chemicals; Chemotaxis; Complex; Cues; Cytoskeletal Modeling; Cytoskeleton; Data; Destinations; Development; Differential Scanning Calorimetry; Down-Regulation; Drosophila genus; Electrons; Epigallocatechin Gallate; Equilibrium; Family; Flavoproteins; Goals; Growth; Growth Cones; Homologous Gene; Hydrogen Peroxide; Immune system; In Vitro; Individual; Injury; Integrins; Intervention; LIM Domain; Life; Ligand Binding; Mediating; Mediator of activation protein; Methods; Mixed Function Oxygenases; Modification; Molecular; Molecular Conformation; Mus; Muscle fasciculation; N-terminal; NADP; Nature; Nerve Regeneration; Neuraxis; Neurons; Neuropilins; Oxidation-Reduction; Oxidoreductase; Oxygen; Phosphotransferases; Physiological; Process; Production; Proline-Rich Domain; Proteins; Reaction; Reactive Oxygen Species; Reducing Agents; Regulation; Reporting; Resolution; Rest; Role; SH3 Domains; Science; Semaphorin-3A; Semaphorins; Shapes; Signal Transduction; Signaling Protein; Skeletal Development; Source; Specific qualifier value; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic Intervention; Titrations; To specify; Transfection; Ultracentrifugation; Vascular Endothelial Growth Factors; Vascular Permeabilities; Vimentin; Work; angiogenesis; axon guidance; axon regeneration; base; calponin; cancer cell; cell motility; design; flavin-containing monooxygenase; follow-up; inhibitor/antagonist; mutant; nerve injury; nervous system development; neurodevelopment; novel; peer; plexin; protein function; 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，轴突通过大量分子提示和排斥信号引导到其最终目的地，这些信号指示细胞骨架重定向生长方向。这些信号之一涉及闪光蛋白与丛蛋白的相互作用。该信号的存在通过另一个分子Mical（与CASL相互作用）传递到细胞骨架上，这是一种具有含FAD的羟化酶（micalfd）结构域的多域蛋白。我们已经确定了MicalFD的结构，并表明它使用NADPH催化了O2至H20 2的降低。 EGCG抑制了这种活性，EGCG是一种单加氧酶抑制剂，它也抑制了信号素的排斥作用。已经表明，Mical在细胞培养中的H20 2产生与细胞收缩相关，这种活性与细胞骨架重组有关。结果表明，H20 2的产生是一个高度调节的过程，涉及FAD结构域与其他微域的相互作用，并通过与Plexin和CRMP的C2结构域的相互作用来控制。此外，已显示Mical与CASL的相互作用已被证明是defastipulation调控的重要组成部分，这是轴突指导中的关键早期过程。该项目的长期目标是使用生物物理方法和原子分辨率3D结构确定来表征微分活性的调节。在这个项目中，我们集中于微分域之间以及mical和CASL之间的相互作用。我们的目的是：1）揭示分子内相互作用自身抑制H20 2产生并调节微分氧化还原活性的机制，以及2）识别和表征Mical与控制Defasticulation的CASL的SH3结构域的相互作用。 轴突指导标志着Playa在中枢神经系统发展和受伤后神经再生中的主要作用。对这些信号负责的相互作用的详细表征对于理解大脑发育以及神经损伤后的药理学干预措施至关重要。