Dissecting new mechanisms of lysosome quality control in health and disease

项目摘要 溶酶体是大分子循环、代谢重组和促生长的关键节点 细胞中的信号。因此，溶酶体功能缺陷是退行性疾病和衰老的基础， 溶酶体的过度活化与癌症有关。先前的研究表明， 胰腺导管腺癌（PDA）细胞上调溶酶体生物发生和活性以促进 降解，清除和回收利用的传入货物材料交付的自噬率增加， 巨胞饮作用是否质的差异赋予PDA溶酶体独特的结构和功能 科普对衬底间隙的更高要求的性能仍然未知。为了回答这个问题，我们 对PDA和正常细胞分离的溶酶体进行了首次比较蛋白质组学分析 并鉴定了膜修复因子Ferlin家族的成员Myoferlin和Dysferlin， 选择性富集在PDA溶酶体的膜上。我们认为Ferlin蛋白赋予 增加对PDA细胞中溶酶体膜应激的保护。 Ferlin蛋白通常定位于受到增强的细胞毒性作用的细胞类型的质膜上。 机械应力，如骨骼肌，在那里它们促进脂质双层的修复。因此，突变 与两种形式的肌营养不良症有关， 损害成肌细胞成熟、融合和质膜修复。因此，我们假设PDA 细胞在溶酶体膜上劫持并重新利用Ferlin蛋白，以保护该细胞器的完整性。在 支持这一假设，我们的初步研究结果表明，PDA溶酶体更耐急性 相对于正常细胞化学诱导的膜透化。从机制上讲，溶酶体定位 的MYOF是必要的，足以维持溶酶体质量控制，其抑制导致 在溶酶体形态、PDA细胞增殖和体内肿瘤生长方面存在严重缺陷。这个目标 研究的目的是研究MYOF如何在机制细节上保护溶酶体膜， 确定阻断溶酶体质量控制对细胞代谢、促生长信号传导和 疾病进展。总之，我们的发现和拟议的研究将是第一个确定一个新的 功能的Ferlin蛋白在溶酶体膜，并提供洞察如何提高溶酶体质量 控制调节细胞内稳态和疾病发病机制。

PROJECT SUMMARY Lysosomes function as critical nodes for macromolecular recycling, metabolic rewiring, and pro-growth signaling in cells. Accordingly, defects in lysosome function underlie degenerative diseases and aging while hyperactivation of lysosomes are associated with cancer. Prior studies have shown that highly aggressive Pancreatic ductal adenocarcinoma (PDA) cells upregulate lysosome biogenesis and activity to facilitate degradation, clearance and recycling of incoming cargo material delivered by increased rates of autophagy and macropinocytosis. Whether qualitative differences endow PDA lysosomes with unique structural and functional properties to cope with a higher demand for substrate clearance remains unknown. To answer this question, we have conducted the first comparative proteomics analysis of lysosomes isolated from PDA versus normal cells and have identified members of the Ferlin family of membrane repair factors, Myoferlin and Dysferlin, as selectively enriched on the membrane of PDA lysosomes. We propose that Ferlin proteins confer increased protection against lysosomal membrane stress in PDA cells. Ferlin proteins are normally localized on the plasma membrane of cell types subjected to heightened mechanical stress, such as skeletal muscle, where they facilitate repair of the lipid bilayer. Accordingly, mutations in DYSF are associated with two forms of muscular dystrophy whereby impaired membrane resealing compromises myoblast maturation, fusion and plasma membrane repair. Therefore, we hypothesize that PDA cells hijack and repurpose Ferlin proteins at the lysosome membrane to protect the integrity of this organelle. In support of this hypothesis, our preliminary findings show that PDA lysosomes are more resistant to acute chemically induced membrane permeabilization relative to normal cells. Mechanistically, lysosome localization of MYOF is necessary and sufficient for maintenance of lysosome quality control and its suppression leads to profound defects in lysosome morphology, PDA cell proliferation and in vivo tumor growth. The goal of this study is to investigate how MYOF functions to protect the lysosome membrane in mechanistic detail and to determine the impact of blocking lysosome quality control on cellular metabolism, pro-growth signaling and disease progression. In summary, our discovery and proposed studies will be the first to determine a novel function for Ferlin proteins at the lysosome membrane and provide insight into how enhanced lysosome quality control regulates cellular homeostasis and disease pathogenesis.