Redox signaling in axon guidance: Structure and activity of MICAL

在神经发育过程中，轴突被大量的分子线索（吸引和排斥信号）引导到它们的最终目的地，这些信号指示细胞骨架重新定向生长方向。这些信号之一涉及脑信号蛋白与丛状蛋白的相互作用。这种信号的存在通过另一种分子MICAL（与CasL相互作用的分子）传递到细胞骨架，MICAL是一种具有含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，并表明它催化使用NADPH将O2还原为H2O2。这种活性被EGCG抑制，EGCG是一种单加氧酶抑制剂，也抑制脑信号蛋白的排斥作用。已经表明，细胞培养物中MICAL产生的H2O2与细胞收缩相关，细胞收缩是与细胞骨架重组相关的活性。结果表明，H2O2的产生是一个高度调节的过程，涉及FAD结构域与其他MICAL结构域的相互作用，并通过与丛状蛋白的C2结构域和CRMP（纤维素反应介体蛋白）的相互作用来控制。此外，MICAL与CasL的相互作用已被证明是调节去纤维化的重要组成部分，这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MICAL活性的调节。在这个项目中，我们集中在MICAL结构域之间的相互作用和MICAL和CasL之间。我们的目标是：1）揭示分子内相互作用自抑制H2O2产生和调节MICAL氧化还原活性的机制，和2）鉴定和表征MICAL与控制去血管化的CasL的SH 3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。负责这些信号的相互作用的详细表征对于理解大脑发育和神经损伤后的药物干预设计至关重要。

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.