Redox signaling in axon guidance: Structure and activity of MICAL

在神经发育期间，轴突被大量的分子线索引导到它们的最终目的地--吸引和排斥的信号，指示细胞骨架改变生长方向。其中一个信号涉及信号素与丛状蛋白的相互作用。这种信号的存在是通过另一种分子Mical(与CASL相互作用的分子)传递到细胞骨架的，Mical是一种含有FAD羟基酶(MICALfd)结构域的多结构域蛋白质。我们测定了MICALfd的结构，结果表明它在NADPH作用下催化O2还原为H202。这种活性被EGCG抑制，EGCG是一种单加氧酶抑制剂，也能抑制信号素的排斥作用。已有研究表明，Mical在细胞培养中产生的H202与细胞收缩有关，细胞收缩与细胞骨架重组有关。结果表明，H202的产生是一个高度调控的过程，涉及FAD结构域和其他Mical结构域的相互作用，并受Plexin的C2结构域和CRMP(崩塌蛋白反应中介蛋白)的相互作用控制。此外，Mical与CASL的相互作用已被证明是轴突引导中一个关键的早期过程--去鞭毛化的调节的重要组成部分。该项目的长期目标是使用生物物理方法和原子分辨率三维结构测定来表征化学活动的规律。在这个项目中，我们主要研究Mical结构域之间以及Mical与CASL之间的相互作用。我们的目标是：1)揭示分子内相互作用抑制H202产生和调节Mical氧化还原活性的机制，以及2)鉴定和表征Mical与CASL的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起着重要作用。对负责这些信号的相互作用的详细描述对于了解大脑发育和设计神经损伤后的药物干预是至关重要的。

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.