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

内溶酶体系统的缺陷与神经退行性变密切相关，内吞细胞器之间正确的蛋白质运输对健康衰老至关重要。囊泡接头蛋白复合物家族（AP-1至AP-5）通过选择货物和协调机器来构建囊泡，充当中央运输控制器。AP-5是最近发现的AP复合体，与后期内体/溶酶体的蛋白质循环有关，并且在饥饿条件下其募集会增强（Hirst等人，2021）。此外，AP-5缺陷细胞具有溶酶体和自噬缺陷，人类AP-5的缺失会导致神经退行性变，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方面发挥作用（Khundadze et al., 2019）。尽管AP-5囊泡通路对神经元健康很重要，但其载货蛋白的定义尚不明确，特别是在应激条件下。该项目的目标是在基础和饥饿条件下鉴定和表征AP-5囊泡货物蛋白，并研究AP-5货物错分类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退行性变的理解。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等人，2018），接近标记（Kubitz等人，2022），CRISPR/ cas9介导的基因编辑，功能细胞生物学分析和高级定量显微镜（Han等人，2020）。您将使用这些方法来鉴定AP-5货物蛋白，可视化细胞中AP-5囊泡运输，并测试是否需要特定的AP-5货物蛋白来维持正常的溶酶体功能和自噬。

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.