Redox signaling in axon guidance: Structure and activity of MICAL

在神经发育过程中，轴突被大量的分子信号引导到它们的最终目的地——吸引和排斥信号，这些信号指示细胞骨架重定向生长方向。其中一个信号涉及信号蛋白与神经丛蛋白的相互作用。该信号通过另一种分子micical（与CasL分子相互作用）传递到细胞骨架，MICALfd是一种多结构域蛋白，具有含有fad的羟化酶（MICALfd）结构域。我们已经确定了MICALfd的结构，并表明它使用NADPH催化O2还原为H20。这种活性被EGCG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，micical在细胞培养中产生的h2o与细胞收缩有关，这是一种与细胞骨架重组相关的活动。结果表明，H20的产生是一个高度调控的过程，涉及FAD结构域与其他micical结构域的相互作用，并受Plexin的C2结构域和CRMP（坍缩反应介质蛋白）的相互作用控制。此外，已确定的micical与CasL的相互作用已被证明是调节去血循环的重要组成部分，去血循环是轴突引导的关键早期过程。该项目的长期目标是使用生物物理方法和原子分辨率3D结构测定来表征micical活性的调节。在这个项目中，我们专注于MICAL领域之间以及MICAL和CasL之间的交互。我们的目标是：1)揭示分子内相互作用自动抑制h2o2产生和调节micical氧化还原活性的机制，2)鉴定和表征micical与CasL的SH3结构域控制去血循环的相互作用。

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.