Determining the Roles of Sphingolipids in Phagocytosis

项目总结 吞噬作用是一种保守的细胞过程，对组织的先天性免疫反应是必不可少的。 动态平衡与多种病理因素有关，包括微生物易感性和自身免疫。 精神错乱。尽管吞噬作用在一个多世纪前就被首次描述，但大多数分子 控制吞噬作用不同阶段的机制，特别是脂-蛋白质相互作用事件，是 人们对此知之甚少。研究表明，吞噬作用涉及复杂的膜重组， 需要在细胞表面进行广泛的脂质重塑。然而，脂类的作用，如鞘脂 在吞噬作用中，Spls的作用目前大多还不清楚。我们假设结构和生物活性的SPL都是 吞噬作用中的关键决定因素，它们参与吞噬细胞突触的信号传递 和/或通过将蛋白质招募到新形成的 吞噬小体。我们最近发现鞘磷脂(SM)的生物合成是人体内主要的SPL之一。 哺乳动物细胞，是吞噬功能的关键。无论SM本身或其生物合成的副产品对 这个过程是未知的。在这项应用中，我们计划表征所需的确切脂质种类 吞噬作用。为此，我们将采取化学和遗传方法来破坏SM的分解代谢 在其降解途径上的点，以检查不同的SM衍生的脂质物种在 吞噬作用。为了深入了解这些脂质在这一过程中发挥作用的机制，我们还计划 确定SPLs在吞噬过程中的定位、代谢转化和相互作用伙伴。为 为此，我们将使用最近报道的遗传编码SM生物传感器以及我们的新的多 功能性SPL前体类似物。这些工具将允许我们检查SPL的亚细胞定位 依赖时间和空间的方式。我们的功能探针将使我们能够识别SPL相互作用的蛋白质 在吞噬的不同阶段。了解这些类脂是如何使 吞噬作用将揭示这一基本的细胞过程，并有助于制定治疗策略 与吞噬细胞紊乱相关的病理学。 8

PROJECT SUMMARY Phagocytosis is a conserved cellular process that is essential for innate immune responses, tissue homeostasis and is associated with various pathologies, including microbial susceptibility and autoimmune disorders. Although phagocytosis was first described more than a century ago, most of the molecular mechanisms that govern the different stages of phagocytosis, especially the lipid-protein interaction events, are poorly understood. Studies have shown that phagocytosis involves complex membrane reorganization that requires extensive remodeling of lipids at the cell surface. However, the roles of lipids, such as sphingolipids (SPLs) in phagocytosis, remain mostly unknown. We hypothesize that both structural and bioactive SPLs are critical determinant factors in phagocytosis where they are involved in signaling at the phagocytic synapse and/or help form the biophysical structure of the phagosome through recruiting proteins to the newly formed phagosomes. We recently found that the biosynthesis of sphingomyelin (SM), one of the major SPLs in mammalian cells, is critical for phagocytosis. Whether SM itself or its biosynthetic byproducts are essential for this process is unknown. In this application, we plan to characterize the exact lipid species required for phagocytosis. To this end, we will take chemical and genetic approaches to disrupt SM catabolism at multiple points along its degradative pathway in order to examine the roles of distinct SM-derived lipid species during phagocytosis. To gain insight into the mechanism by which these lipids play roles in this process, we also plan to determine the localization, metabolic conversions, and interacting partners of SPLs during phagocytosis. For this, we will employ the recently reported genetically encoded SM biosensor as well as our novel multi- functional SPL precursor analogs. These tools will allow us to examine the subcellular localization of SPLs in a time- and space-dependent manner. Our functional probes will enable us to identify SPL interacting proteins during the different stages of phagocytosis. An understanding of how these class of lipids enables phagocytosis will reveal insights into this fundamental cellular process and help develop therapeutic strategies to pathologies related to phagocytic disorders. 8