Lysosomal quality control through lipid remodeling

项目摘要/摘要： 溶酶体在细胞生理学中起着至关重要的作用，不仅控制营养循环和细胞生长，而且 也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 以及许多疾病，如溶酶体储存病、神经退行性变和心血管疾病。一个 溶酶体相关疾病的标志是溶酶体膜通透性/损伤(LMP)，如果不是 立即解决可能会导致包括细胞死亡在内的有害问题。我们现在开始理解LMP 触发多种细胞途径修复受损的溶酶体。然而，前面描述的 这些途径似乎对溶酶体的快速修复是必不可少的，这暗示了其他修复机制。作为一种 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屏幕搜索 专门浓缩在受损溶酶体上的蛋白质。这一筛选导致了对 磷脂酰肌醇启动的膜拴系和脂质转运(PITT)途径是一种重要机制 用于快速修复溶酶体。我们发现LMP刺激磷脂酰肌醇-4-磷酸的强劲生产 (PtdIns4P，PI4P)对II型磷脂酰肌醇-4激酶(PI4K2A)损伤的溶酶体的影响。溶酶体 PI4P促使内质网和内质网之间形成广泛的膜接触 招募多个氧固醇结合蛋白相关蛋白(ORP)家族对溶酶体的损伤 会员。ORPs催化胆固醇和磷脂酰丝氨酸随后从内质网到溶酶体的运输 (PS)介导膜的快速修复。虽然胆固醇本身增加了细胞膜的稳定性，但PS激活了 ATG2介导的直接溶酶体修复的脂类转运。PITT通路被激活，以响应不同的 与疾病相关的溶酶体破坏性条件，预计将对人类产生巨大影响 病理生理学。值得注意的是，皮特途径不仅揭示了膜接触处的脂质转移 溶酶体修复的基本机制，但它也建立了脂质重塑作为一个新的平台 了解溶酶体的质量控制。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，以更好地从机制上理解溶酶体 质量控制和潜在的治疗应用。首先，我们在提纯溶酶体期间和之后 溶酶体修复以脂类组学表征脂质变化，我们相信这将识别新的脂类信使 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 一个研究胆固醇运输的很好的细胞模型，我们特别感兴趣的是 胆固醇从新修复的溶酶体中排出。最后，我们还使用FDA进行化学筛查- 已批准的化学库用于搜索激活或阻断PITT途径的小分子。被识别的人 小分子已经很好地建立了蛋白质靶标，这将有助于定义PIT的调控网络 溶酶体质量控制途径以及描述提高溶酶体质量的新策略。

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.