Determining the Roles of Sphingolipids in Phagocytosis

项目摘要 吞噬作用是一种保守的细胞过程，对于先天性免疫应答、组织免疫应答和细胞免疫应答是必不可少的。 体内平衡，并与各种病理学，包括微生物易感性和自身免疫性 紊乱虽然吞噬作用是在世纪前首次描述的，但大多数分子 控制吞噬作用的不同阶段，特别是脂质-蛋白质相互作用事件的机制是 不太了解。研究表明，吞噬作用涉及复杂的膜重组， 需要细胞表面脂质的广泛重塑。然而，脂质，如鞘脂， （SPLs）在吞噬作用中的作用，大部分仍然未知。我们假设结构和生物活性SPL都是 吞噬作用中的关键决定因子，参与吞噬突触的信号传导 和/或通过向新形成的吞噬体募集蛋白质来帮助形成吞噬体的生物物理结构 吞噬体我们最近发现，鞘磷脂（SM）的生物合成，其中一个主要的SPLs在 哺乳动物细胞，是吞噬作用的关键。SM本身或其生物合成副产物是否对 这个过程是未知的。在本申请中，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 catalysts在多个 点沿着其降解途径，以检查不同的SM衍生的脂质种类的作用， 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 以确定定位，代谢转换，和相互作用的配偶体的SPLs在吞噬作用。为 为此，我们将采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。这些工具将使我们能够检查SPL的亚细胞定位， 时间和空间依赖的方式。我们的功能探针将使我们能够识别SPL相互作用蛋白 在吞噬作用的不同阶段。了解这类脂质如何使 吞噬作用将揭示对这一基本细胞过程的认识，并有助于开发治疗策略 与吞噬细胞疾病有关的病理学。 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