Lysosomal quality control through lipid remodeling

项目概要/摘要： 溶酶体在细胞生理学中发挥重要作用，不仅控制营养循环和细胞生长， 还调节各种细胞应激的适当处理。溶酶体功能障碍与衰老有关 和许多疾病如溶酶体贮积病、神经变性和心血管疾病。一 溶酶体相关疾病的标志是溶酶体膜透化/损伤（LMP）， 立即解决可能导致有害的问题，包括细胞死亡。我们现在开始了解LMP 触发多种细胞通路修复受损的溶酶体然而，先前描述的任何一个都不 途径似乎是必需的快速溶酶体修复，提示额外的修复机制。作为 为了寻找这种机制，我们最近设计并执行了一个无偏的蛋白质组筛选搜索 在受损的溶酶体上富集的蛋白质。这个屏幕导致了 磷酸肌醇启动的膜束缚和脂质转运（PITT）途径是一种重要机制 快速溶酶体修复我们发现，LMP刺激磷脂酰肌醇-4-磷酸的强劲生产 （PtdIns 4P，PI 4P）通过II型α磷脂酰肌醇-4激酶（PI 4K 2A）作用于受损的溶酶体。溶酶体 PI 4P驱动内质网（ER）和内质网（ER）之间广泛膜接触的形成。 通过募集多个氧固醇结合蛋白（OSBP）相关蛋白（ORP）家族而损伤溶酶体 成员氧化还原酶催化胆固醇和磷脂酰丝氨酸的内质网向溶酶体的转运 (PS)介导快速膜修复。虽然胆固醇本身增加膜的稳定性，PS激活 用于直接溶酶体修复的ATG 2介导的脂质转运。PITT通路是在对不同的 疾病相关的溶酶体损伤条件，并预计将对人类产生巨大的影响 病理生理学值得注意的是，PITT途径不仅揭示了膜接触处的脂质转移， 溶酶体修复的重要机制，但它也建立了脂质重塑作为一个新的平台， 了解溶酶体质量控制。通过未来五年的三个独立项目，我们的实验室将 继续研究LMP触发的溶酶体脂质重塑，以更好地了解溶酶体脂质重塑的机制。 质量控制和潜在的治疗应用。首先，我们在纯化期间和之后的溶酶体 溶酶体修复来表征脂质变化的脂质组学，我们相信这将确定新的脂质信使 对溶酶体质量控制很重要。其次，PITT介导的溶酶体胆固醇积累提供了 一个很好的细胞模型来研究胆固醇的运输，我们特别感兴趣的机制， 胆固醇从新修复的溶酶体排出。最后，我们还使用FDA进行化学筛选- 该实验室使用经批准的化学库来搜索激活或阻断PITT途径的小分子。所识别的 小分子已经建立了很好的蛋白质靶点，这将有助于确定PITT的调控网络 溶酶体质量控制途径以及描绘新的策略，以改善溶酶体质量。

Project Summary/Abstract: Lysosomes play essential roles in cell physiology, not only controlling nutrient recycling and cellular growth, but also mediating the proper handling of various cellular stress. Lysosomal dysfunction is associated with aging and many diseases such as lysosomal storage disease, neurodegeneration, and cardiovascular diseases. A hallmark of lysosomal-related diseases is lysosomal membrane permeabilization/damage (LMP) which if not immediately resolved can cause detrimental problems including cell death. We now start to understand that LMP triggers multiple cellular pathways to repair damaged lysosomes. However, none of the previously described pathways appear to be essential for rapid lysosomal repair, suggesting additional repair mechanisms. As an attempt to find such mechanism, we recently designed and executed an unbiased proteomic screen searching for proteins specifically enriched on damaged lysosomes. This screen led to the discovery of the phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway as an essential mechanism for rapid lysosomal repair. We found that LMP stimulates robust production of phosphatidylinositol-4-phosphate (PtdIns4P, PI4P) on damaged lysosomes by type II alpha phosphatidylinositol-4 kinase (PI4K2A). Lysosomal PI4P drives the formation of extensive membrane contacts between the endoplasmic reticulum (ER) and damaged lysosomes by recruiting multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members. The ORPs catalyze subsequent ER-to-lysosomal transport of cholesterol and phosphatidylserine (PS) to mediate rapid membrane repair. While cholesterol by itself increases membrane stability, PS activates ATG2-mediated lipid transport for direct lysosomal repair. The PITT pathway is activated in response to diverse disease-related lysosomal-damaging conditions and is expected to have enormous impact on human pathophysiology. Remarkably, the PITT pathway not only reveals lipid transfer at membrane contacts as a essential mechanism for lysosomal repair, but it also establishes lipid remodeling as a new platform to understand lysosomal quality control. Through three independent projects in the next five years, our lab will continue studying LMP-triggered lysosomal lipid remodeling for better mechanistic understanding of lysosomal quality control and potential therapeutic applications. First, we are purifying lysosomes during and after lysosomal repair to characterize lipid changes by lipidomics, which we believe will identify new lipid messengers important for lysosomal quality control. Second, the PITT-mediated lysosomal cholesterol accumulation provides a great cellular model to study cholesterol transport, and we are particularly interested in the mechanism for cholesterol egress from newly repaired lysosomes. Finally, we are also performing chemical screens using FDA- approved chemical library to search for small molecules that activate or block the PITT pathway. The identified small molecules have well established protein targets, which will help define the regulatory networks for the PITT lysosomal quality control pathway as well as delineate new strategies to improve lysosomal quality.