Regulation and Function of Phosphoinositide Lipid Signals

磷酸肌醇脂质信号的调节和功能

• 批准号: RGPIN-2020-04343
• 负责人: Botelho, Roberto
• 金额: $ 4.23万
• 依托单位: Ryerson University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739904
• 关键词: Regulation; Function; Phosphoinositide; Lipid; Signals

Organelles are the "organs" of cells. There are many types of organelles, each with their own unique biochemical and functional properties. For example, the endoplasmic reticulum (ER) is a labyrinth of membrane tubules where protein synthesis occurs, whereas lysosomes are small, round organelles packed with digestive enzymes that eliminate unwanted materials like damaged proteins and microbes. How organelles form, or change, is a key question in cell biology. The phosphoinositide (PIP) lipids are architects of organelle identity. There are seven PIP species that are differentially distributed among organelles. Each PIP species binds a unique set of proteins, which decorates the host organelle with specific molecular properties. In order to understand how PIPs define organelle identity, we need to understand A) how the enzymes that synthesize and degrade PIPs are regulated and B) how PIPs and their effector proteins work. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is a specific PIP that is synthesized by the lipid kinase PIKfyve. PI(3,5)P2 governs lysosome properties, which is important for cells to clear unwanted material. My NSERC-funded research has focused on understanding how PIKfyve is regulated and how it functions. We showed that inhibition of PIKfyve impairs immune function, including neutrophil migration towards microbes (chemotaxis) and the engulfment and digestion of microbes (phagocytosis and phagosome maturation). In addition, we discovered that lysosomes fail to separate (fission) from other lysosomes after fusion during PIKfyve ablation, causing their coalescence. This separation defect may occur because PIKfyve modulates protein machinery that generate force and/or deforms membranes that elicit fission. With NSERC support: i)We will use unbiased methods based on lysosome isolation, molecular tagging, and mass spectrometry to understand how the composition of lysosomes change during PIKfyve ablation. These data may identify complexes involved in membrane fission. ii)We will study the mechanisms by which PIKfyve governs phagocytosis and cell migration. We will use immune and cancer cells to test the hypothesis that PIKfyve coordinates the assembly and disassembly of the actin cytoskeleton to mold cell shape and generate forces necessary for these processes. iii)We will study how PIKfyve modulates the assembly of actin on lysosomes and if this interfaces with the ER to demarcate fission sites. ER contact sites with other organelles is emerging as a mediator of organelle fission. In all, this research will provide new insight into how PIKfyve governs lysosome function and interfaces with the cell's force generating machinery. This may then aid researchers better understand deleterious effects caused by PIKfyve loss. In turn, this may provide the Canadian pharmaceutical industry with novel strategies to treat conditions like Charcot-Marie Tooth neurodegenerative disease caused by PI(3,5)P2 malfunction.

细胞器是细胞的“器官”。细胞器有多种类型，每种都有其独特的生化和功能特性。例如，内质网（ER）是一个膜管迷宫，蛋白质合成发生在其中，而溶酶体是小型圆形细胞器，充满消化酶，可以消除不需要的物质，如受损的蛋白质和微生物。细胞器如何形成或变化是细胞生物学的一个关键问题。磷酸肌醇 (PIP) 脂质是细胞器特性的建筑师。有七种 PIP 物种，它们在细胞器中的分布存在差异。每个 PIP 物种都结合一组独特的蛋白质，这些蛋白质用特定的分子特性装饰宿主细胞器。为了了解 PIP 如何定义细胞器特性，我们需要了解 A) 合成和降解 PIP 的酶如何受到调节，以及 B) PIP 及其效应蛋白如何发挥作用。磷脂酰肌醇-3,5-二磷酸 [PI(3,5)P2] 是一种由脂质激酶 PIKfyve 合成的特定 PIP。 PI(3,5)P2 控制溶酶体特性，这对于细胞清除不需要的物质非常重要。我的 NSERC 资助的研究重点是了解 PIKfyve 的监管方式及其运作方式。我们发现，抑制 PIKfyve 会损害免疫功能，包括中性粒细胞向微生物迁移（趋化性）以及微生物的吞噬和消化（吞噬作用和吞噬体成熟）。此外，我们发现在 PIKfyve 消融过程中，溶酶体在融合后无法与其他溶酶体分离（裂变），导致它们聚结。这种分离缺陷的发生可能是因为 PIKfyve 调节产生力和/或使膜变形从而引发裂变的蛋白质机制。在 NSERC 的支持下： i) 我们将使用基于溶酶体分离、分子标记和质谱的无偏方法来了解 PIKfyve 消融过程中溶酶体的组成如何变化。这些数据可以识别参与膜裂变的复合物。 ii)我们将研究 PIKfyve 控制吞噬作用和细胞迁移的机制。我们将使用免疫细胞和癌细胞来检验以下假设：PIKfyve 协调肌动蛋白细胞骨架的组装和分解，以塑造细胞形状并产生这些过程所需的力。 iii)我们将研究 PIKfyve 如何调节肌动蛋白在溶酶体上的组装，以及它是否与 ER 相互作用来划分裂变位点。内质网与其他细胞器的接触位点正在成为细胞器裂变的介质。 总之，这项研究将为 PIKfyve 如何控制溶酶体功能以及如何与细胞的力产生机制相互作用提供新的见解。这可能有助于研究人员更好地了解 PIKfyve 丢失造成的有害影响。反过来，这可能为加拿大制药业提供新的策略来治疗由 PI(3,5)P2 功能障碍引起的腓骨肌萎缩症等神经退行性疾病。