Regulatory role of the ubiquitin ligase Itch in endocytosis and synaptic vesicle recycling

泛素连接酶 Itch 在内吞作用和突触小泡回收中的调节作用

• 批准号: RGPIN-2017-05054
• 负责人: Angers, Annie
• 金额: $ 1.89万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710438
• 关键词: Regulatory; role; ubiquitin; ligase; Itch

Endocytosis is a fundamental cell process in which a cell absorbs a portion of its membrane as well as the proteins associated to it. This phenomenon plays a crucial role in the regulation of intracellular signalling by controlling the amount of cell-surface receptors. It is also closely linked to the maintenance of neuronal activity. When neurons are firing, excitatory neurotransmitters are released at the synapse by fusion of synaptic vesicles with the synaptic membrane. These vesicles are filled with neurotransmitters that are freed into the intercellular space upon fusion of the synaptic vesicles with the cell membrane. To ensure the maintenance of the membrane and of the synaptic vesicle pools, synaptic vesicles as well as their associated proteins must be recovered from the cell membrane by endocytosis. The molecular mechanisms controlling receptor internalization and synaptic vesicle recycling are very similar, and the regulatory proteins involved in both processes are the same. Endophilin is one of the proteins governing endocytosis and is essential for synaptic vesicle recycling. We discovered that endophilin is targeted by an enzyme called Itch that modifies endophilin by fusing it to ubiquitin, which is a signal for protein degradation. When we knocked down Itch in developing zebrafish embryos, the development was perturbed due to the misregulation of growth factor receptors, leading to an excess of their target genes and developmental defects; however, Endophilin is not the only target of Itch's activity, and it is not known how Itch really affects these receptors. Our research program is dedicated to the elucidation of regulatory mechanisms of synaptic vesicle recycling. To establish the contribution of Itch and Endophilin in this process, we propose to specifically interfere with the interaction between Itch and Endophilin, and thereby establish a potentially new regulatory mechanism of endocytosis and synaptic vesicle recycling. We will genetically alter somatic and neuronal cells to modify the gene coding the Itch protein in a way that it will no longer be able to recognize and modify endophilin, while leaving its ability to modify other substrates intact. We expect that blocking the interaction between Itch and endophilin will result in increased endophilin levels, perturbing receptor internalization and synaptic vesicle recycling. We will use different molecular and cell imaging techniques to measure the extent of these perturbations. These experiments will provide important fundamental knowledge on the fine-tuning of crucial processes in the maintenance of cell function and maybe provide new avenues in research for the treatment of cellular diseases such as cancer and neurodegeneration.

内吞作用是细胞吸收部分细胞膜及其相关蛋白质的基本过程，通过控制细胞表面受体的数量，在细胞内信号转导的调节中起着至关重要的作用。它也与神经元活动的维持密切相关。当神经元放电时，兴奋性神经递质通过突触囊泡与突触膜的融合在突触处释放。这些囊泡充满了神经递质，当突触囊泡与细胞膜融合时，这些神经递质被释放到细胞间隙中。为了确保膜和突触囊泡池的维持，必须通过胞吞作用从细胞膜回收突触囊泡及其相关蛋白。控制受体内化和突触囊泡回收的分子机制非常相似，参与这两个过程的调节蛋白也是相同的。嗜内蛋白是一种控制内吞作用的蛋白质，对突触小泡的再循环至关重要。我们发现，内啡肽是由一种称为Itch的酶靶向的，该酶通过将内啡肽与泛素融合来修饰内啡肽，泛素是蛋白质降解的信号。当我们在发育中的斑马鱼胚胎中敲除Itch时，由于生长因子受体的错误调节，发育受到干扰，导致其靶基因过量和发育缺陷;然而，Endophilin不是Itch活性的唯一靶标，目前还不知道Itch如何真正影响这些受体。我们的研究项目致力于阐明突触囊泡再循环的调控机制。为了确定Itch和Endophilin在这一过程中的作用，我们建议特异性地干扰Itch和Endophilin之间的相互作用，从而建立一个潜在的新的内吞作用和突触囊泡再循环的调节机制。我们将从基因上改变体细胞和神经元细胞，以某种方式修改编码Itch蛋白的基因，使其不再能够识别和修饰内啡肽，同时保持其修饰其他底物的能力不变。我们预期阻断瘙痒和内亲素之间的相互作用将导致内亲素水平增加，干扰受体内化和突触囊泡再循环。我们将使用不同的分子和细胞成像技术来测量这些扰动的程度。这些实验将为细胞功能维持中关键过程的微调提供重要的基础知识，并可能为治疗癌症和神经变性等细胞疾病的研究提供新的途径。