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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748528
• 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”。有许多类型的细胞器，每种都有自己独特的生化和功能特性。例如，内质网（ER）是蛋白质合成发生的膜小管迷宫，而溶酶体是小而圆的细胞器，充满了消化酶，可以消除不需要的物质，如受损的蛋白质和微生物。细胞器如何形成或改变是细胞生物学中的一个关键问题。磷脂酰肌醇（PIP）脂质是细胞器身份的建筑师。有七种PIP物种在细胞器中有差异分布。每个PIP种类都结合了一组独特的蛋白质，这些蛋白质用特定的分子特性装饰宿主细胞器。为了理解PIP如何定义细胞器的身份，我们需要了解A）合成和降解PIP的酶是如何被调节的，以及B）PIP及其效应蛋白是如何工作的。磷脂酰肌醇-3，5-二磷酸[PI（3，5）P2]是由脂质激酶PIKfyve合成的特异性PIP。PI（3，5）P2控制溶酶体性质，这对于细胞清除不需要的物质是重要的。我的NSERC资助的研究重点是了解PIKfyve是如何监管的，以及它是如何运作的。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的迁移（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消融过程中融合后，溶酶体无法与其他溶酶体分离（分裂），导致它们聚结。这种分离缺陷可能发生，因为PIKfyve调节蛋白质机制，产生力和/或使引起裂变的膜变形。NSERC支持：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消融期间溶酶体组成的变化。这些数据可以确定涉及膜分裂的复合物。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测试PIKfyve协调肌动蛋白细胞骨架的组装和拆卸以塑造细胞形状并产生这些过程所需的力的假设。iii）我们将研究PIKfyve如何调节肌动蛋白在溶酶体上的组装，以及这是否与ER相互作用以划分裂变位点。内质网与其他细胞器的接触部位正在成为细胞器裂变的媒介。 总之，这项研究将为PIKfyve如何控制溶酶体功能以及与细胞的力产生机制的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损失造成的有害影响。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Charcot-Marie Tooth神经退行性疾病等疾病。