Advanced proteomics and microscopy to investigate protein trafficking in the endolysosomal system and its links with neurodegeneration

先进的蛋白质组学和显微镜技术研究内溶酶体系统中的蛋白质运输及其与神经退行性变的联系

• 批准号: 2898732
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2898732
• 关键词: Advanced; proteomics; microscopy; investigate; protein

Defects in the endolysosomal system are strongly linked to neurodegeneration and correct trafficking of proteins between endocytic organelles is essential for healthy ageing. The adaptor protein complex family of vesicle adaptors (AP-1 to AP-5) act as central trafficking controllers, by selecting cargo and coordinating machinery to build vesicles. AP-5, the most recently discovered AP complex, is implicated in protein recycling at late endosomes/lysosomes and its recruitment is enhanced under starvation conditions (Hirst et al., 2021). Furthermore, AP-5-deficient cells have lysosomal and autophagy defects, and loss of AP-5 in humans leads to neurodegeneration, suggesting that AP-5 may play a role in protecting neurons from stress- or age-induced damage (Khundadze et al., 2019). Despite its importance for neuronal health, the cargo proteins of the AP-5 vesicle pathway are poorly defined, particularly under stress conditions.The goal of this project is to identify and characterise AP-5 vesicle cargo proteins under basal and starvation conditions and to investigate the role of AP-5 cargo missorting in lysosomal and autophagic dysfunction. Ultimately, this will further our understanding of how protein trafficking in the endolysosomal system maintains a healthy cell and how defects in this pathway lead to neurodegeneration.You will be trained in cutting-edge proteomics and cell biology approaches, including mass spectrometry-based spatial proteomics (Davies et al., 2018), proximity labelling (Kubitz et al., 2022), CRISPR/Cas9-mediated gene editing, functional cell biology assays, and advanced quantitative microscopy (Han et al., 2020). You will use these methods to identify AP-5 cargo proteins, visualise AP-5 vesicle trafficking in cells and test whether specific AP-5 cargo proteins are required for normal lysosomal function and autophagy.

内溶酶体系统的缺陷与神经变性密切相关，内吞细胞器之间蛋白质的正确运输对于健康衰老至关重要。囊泡衔接子的衔接蛋白复合物家族（AP-1至AP-5）通过选择货物和协调机制来构建囊泡，从而充当中央运输控制器。AP-5是最近发现的AP复合物，涉及晚期内体/溶酶体的蛋白质再循环，并且其募集在饥饿条件下增强（Hirst et al.，2021年）。此外，AP-5缺陷细胞具有溶酶体和自噬缺陷，并且人类中AP-5的缺失导致神经变性，这表明AP-5可能在保护神经元免受应激或年龄诱导的损伤中起作用（Khundadze et al. 2019年）。尽管AP-5囊泡通路对神经元健康很重要，但其货物蛋白的定义很不明确，特别是在应激条件下，本项目的目标是在基础和饥饿条件下识别和定位AP-5囊泡货物蛋白，并研究AP-5货物错配在溶酶体和自噬功能障碍中的作用。最终，这将进一步加深我们对内溶酶体系统中蛋白质运输如何维持健康细胞以及该途径中的缺陷如何导致神经变性的理解。您将接受尖端蛋白质组学和细胞生物学方法的培训，包括基于质谱的空间蛋白质组学（Davies et al.，2018）、邻近标记（Kubitz等人，2022）、CRISPR/Cas9介导的基因编辑、功能性细胞生物学测定和先进的定量显微术（Han等人，2020年）。您将使用这些方法来识别AP-5货物蛋白，可视化AP-5囊泡在细胞中的运输，并测试特定的AP-5货物蛋白是否是正常溶酶体功能和自噬所必需的。