Molecular analysis of the chaperone Hsc70-Trio protein complex in the neuronal growth cone

神经元生长锥中伴侣 Hsc70-Trio 蛋白复合物的分子分析

• 批准号: RGPIN-2017-04809
• 负责人: LamarcheVane, Nathalie
• 金额: $ 2.04万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682585
• 关键词: Molecular; analysis; chaperone; Hsc70; Trio

During development, the pathfinding of neurons is governed by guidance cues, such as the secreted protein netrin-1. Binding of netrin-1 to the receptor Deleted in Colorectal Cancer (DCC) expressed at the axon's distal tip, the neuronal growth cone, initiates molecular signals that modulate and change the cell cytoskeleton dynamics, leading to axon outgrowth and guidance. We identified the guanine nucleotide exchange factor (GEF) Trio as a key component of netrin-1/DCC signaling in growth cone guidance. Trio is a large (330kDa) cytoplasmic protein containing three catalytic domains, including a serine/threonine kinase domain and two GEFD1 and GEFD2 acting as activators of Rac1 and RhoA, respectively. Trio contains other motifs, including a Sec14 lipid-binding domain, eight spectrin repeats, two Src Homology 3 domains, and an immunoglobulin-like domain. The presence of these various domains suggests that Trio functions as an integrator of multiple upstream inputs and an activator of multiple downstream pathways. However, understanding how Trio integrates these signals is limited by the lack of knowledge in the structure of the protein. To characterize the mechanisms governing Trio localization and activation downstream of netrin-1/DCC, we employed a proteomic approach and identified the molecular chaperone Hsc70 as a novel Trio regulator in the developing cortex. In addition, we identified four Trio phosphorylated serine residues for which the exact function still remains to be elucidated. ***Overall, our recent work highlighted an important role for Trio in neurodevelopment. However, the molecular mechanisms by which Trio modulates the response of axons to guidance cues still remain elusive. Therefore, the goal of this research program is to investigate the regulation, localization, and molecular structure of Trio protein complexes at the neuronal growth cone, in three specific aims:***Aim 1: To characterize the role of serine phosphorylation in Trio function and regulation ***Aim 2: To identify the global Trio interactome in neurite outgrowth using a BioID approach***Aim 3. To elucidate the molecular structure of the Hsc70-Trio protein complex at growth cones using high-resolution molecular cryo-electron microscopy (cryo-EM) combined with live cell imaging using fluorescence microscopy (correlative light electron microscopy or CLEM).***These studies will provide the structural basis for understanding the molecular functions of Trio protein complexes assembled at the leading edge of neuronal growth cones. The interdisciplinary nature of this proposal will give the students the opportunity of learning a wide range of molecular and cellular techniques to analyze protein complexes in neurodevelopment. The current proposal fits within the long-term goals of my program, which are to increase the knowledge of fundamental cytoskeletal-dependent mechanisms during development.

在发育过程中，神经元的寻路是由指导线索，如分泌蛋白netrin-1。netrin-1与在轴突的远端尖端（神经元生长锥）表达的结直肠癌（DCC）中的受体p53的结合启动调节和改变细胞细胞骨架动力学的分子信号，导致轴突生长和引导。我们确定了鸟嘌呤核苷酸交换因子（GEF）Trio作为生长锥引导中netrin-1/DCC信号传导的关键组分。Trio是一种大的（330 kDa）细胞质蛋白，含有三个催化结构域，包括一个丝氨酸/苏氨酸激酶结构域和两个分别充当Rac 1和RhoA激活剂的GEFD 1和GEFD 2。Trio包含其他基序，包括Sec 14脂质结合结构域，8个血影蛋白重复序列，2个Src同源3结构域和1个免疫球蛋白样结构域。这些不同结构域的存在表明，Trio作为多个上游输入的整合剂和多个下游途径的激活剂发挥作用。然而，了解Trio如何整合这些信号受到缺乏蛋白质结构知识的限制。为了表征三重定位和netrin-1/DCC下游激活的机制，我们采用了蛋白质组学方法，并确定了分子伴侣Hsc 70作为一种新的三重调节器在发育中的皮质。此外，我们确定了四个三磷酸化丝氨酸残基的确切功能仍有待阐明。* 总的来说，我们最近的工作强调了Trio在神经发育中的重要作用。然而，Trio调节轴突对引导信号的反应的分子机制仍然难以捉摸。因此，本研究计划的目标是研究Trio蛋白复合物在神经元生长锥的调节、定位和分子结构，有三个具体目标：* 目标1：表征丝氨酸磷酸化在Trio功能和调节中的作用 * 目标2：使用BioID方法鉴定神经突生长中的整体Trio相互作用组 * 目标3。 使用高分辨率分子冷冻电子显微镜（cryo-EM）结合使用荧光显微镜（相关光电子显微镜或CLEM）的活细胞成像，阐明生长锥中Hsc 70-Trio蛋白复合物的分子结构。*这些研究将为理解在神经生长锥前缘组装的Trio蛋白复合物的分子功能提供结构基础。该提案的跨学科性质将使学生有机会学习广泛的分子和细胞技术，以分析神经发育中的蛋白质复合物。目前的提议符合我的计划的长期目标，即增加对发育过程中基本细胞因子依赖机制的了解。