Phosphatidylinositol phosphates and their effector proteins in phagosome-lysosome tethering and fusion

磷脂酰肌醇磷酸盐及其在吞噬体-溶酶体束缚和融合中的效应蛋白

• 批准号: 414783339
• 负责人: Professor Dr. Albert Haas
• 金额: --
• 依托单位: Institut für Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/414783339?language=en
• 关键词: Phosphatidylinositol; phosphates; their; effector; proteins

Phagocytosis is a specialized form of endocytosis during which particles are ingested and delivered to phagolysosomes for degradation. Phagosomes develop into phagolysosomes as they sequentially fuse with early endosomes, late endosomes and lysosomes. To this end, phagosomes and endosomes/lysosomes need to move toward each other and to bind cognate sets of fusion factors. Small GTPases of the Rab- and Arf-families guide phagosome transport and fusion by anchoring motor proteins and fusion factors to phagosome membranes. Fusion between phagosomes and late endosomes or lysosomes depends on HOPS, a multi-subunit tethering complex which bridges compartment-specific Rab- and/or Arf-GTPases before fusion. Many proteins bind to HOPS and may link it to membranes. However, it is not clear which HOPS interactors are bridged during which fusion step of phagosome maturation. The proposed study will analyze the various fusion steps along phagosome maturation for a participation of HOPS and interacting proteins (i.e., Rab2a, Rab7a, Arl8, BORC, and SKIP) to determine which proteins cooperate with HOPS at which stage of the pathway. Additionally, the project will investigate how phosphatidylinositol phosphate lipids (PIPs) and the PIP-binding HOPS interactor SKIP contribute to assembly and membrane binding of HOPS. In particular, a biochemical assay that reconstitutes fusion of purified phagosomes and endosomes or lysosomes will be employed to determine the contributions of HOPS and its various interactors to fusion. This assay not only allows to identify fusion factors but also to assign the action of fusion factors to a specific sub-reaction of fusion, such as tethering. The project will further combine reconstituted stage-specific fusion reactions with co-immunoprecipitation and mass spectrometry to identify which proteins bind to HOPS during which fusion step of phagosome maturation in an unbiased fashion. Centering on phagocytosis, a central part of innate immunity, this study will not only help to understand the biochemistry of membrane fusion but will also provide target information for drug development, in particular against intracellular pathogens.

吞噬作用是一种特殊形式的内吞作用，在此过程中，颗粒被摄取并输送到吞噬小体进行降解。吞噬小体在与早期内小体、晚期内小体和溶酶体依次融合时发育成吞噬小体。为此，吞噬小体和内小体/溶酶体需要相互移动，并结合同源的融合因子集。Rab和Arf家族的小GTP酶通过将运动蛋白和融合因子锚定在吞噬体膜上来指导吞噬小体的运输和融合。吞噬小体与晚期内小体或溶酶体之间的融合依赖于HOP，这是一种多亚基拴系复合体，在融合前连接特定隔室的Rab和/或Arf-GTP酶。许多蛋白质结合到啤酒花上，并可能将其连接到膜上。然而，目前还不清楚在吞噬体成熟的哪个融合步骤中，哪些啤酒花相互作用子被桥接。这项拟议的研究将分析吞噬小体成熟过程中参与啤酒花和相互作用蛋白(即Rab2a、Rab7a、Ar18、BORC和SKIP)的不同融合步骤，以确定哪些蛋白质在途径的哪个阶段与啤酒花合作。此外，该项目还将调查磷脂酰肌醇磷酸脂(PIP)和PIP结合啤酒花相互作用元件SKIP如何促进啤酒花的组装和膜结合。特别是，重组纯化的吞噬小体和内噬菌体或溶酶体融合的生化分析将被用来确定啤酒花及其各种相互作用因子对融合的贡献。这种检测不仅可以识别融合因子，还可以将融合因子的作用分配给特定的融合子反应，如系留。该项目将进一步将重组的阶段特异性融合反应与免疫共沉淀和质谱学相结合，以公正的方式识别在吞噬小体成熟的哪个融合步骤中哪些蛋白质与啤酒花结合。围绕天然免疫的核心部分吞噬作用，本研究不仅有助于了解膜融合的生物化学，而且将为药物开发，特别是针对细胞内病原体的药物开发提供靶向信息。