Role of PTEN during Pi(4,5)P2 homeostasis and autophagy

PTEN 在 Pi(4,5)P2 稳态和自噬中的作用

• 批准号: RGPIN-2017-05170
• 负责人: Carréno, Sébastien
• 金额: $ 1.89万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=646910
• 关键词: Role; PTEN; during; Pi; P2

Autophagy, the process of self-eating, is evolutionarily conserved from yeast to mammals. This process leads to the lysosomal degradation of cytosolic components such as organelles, proteins or nucleic acids. Autophagy starts with the formation of a double-membrane sac that expends and forms the autophagosome that can engulf cytosolic components. The autophagosome then fuse with lysosomes that discharge their digestive enzymes to promote cargo degradation. Autophagy regulates the dynamic remodeling of subcellular compartments and participates to the control of several physiological processes such as embryogenesis, response to starvation, anti-tumorigenesis and anti-senescence. Therefore, the understanding of this important process is essential for both fundamental and biomedical research.****We conducted an in cellulo Drosophila screen on candidate genes controlling the phosphoinositide cycle and we found that the inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis (Ben El Kadhi et al., Current biology 2011). The majority of the PI(4,5)P2 is concentrated at the plasma membrane where it participates in nearly all events that involve the cell surface. We demonstrated that dOCRL is associated with endosomes and lysosomes and that it dephosphorylates PI(4,5)P2 on lysosomes to restrict this phosphoinositide at the plasma membrane. When dOCRL is knocked-down by RNAi, cells abnormally accumulate PI(4,5)P2 at the surface of giant lysosomes. In addition, we recently reported that PTEN activation promotes the hydrolysis of PI(4,5)P2, its own enzymatic product, by activating Phospholipase C (PLC) enzymes on endomembranes. We showed that this function can rescue dOCRL loss (Ben El Kadhi et al., in prep).****In our unpublished observations we showed that depletion of dOCRL leads to a defect in the autophagic flux. Lysosomes cannot longer fuse with autophagosome. Importantly we showed that activation PTEN activation can restore the autophagic flux in dOCRL depleted cells. We also discovered that this function was independent of PTEN enzymatic activity but was supported by a minimal chimera encompassing two of its non-enzymatic conserved domains. In addition we found that PTEN depletion promotes accumulation of Pi(4,5)P2 on endomembranes. ***Besides its canonical role in autophagy activation by inhibiting the PI3K/Akt/mTORC1 pathway, our results suggest a novel role of PTEN in controlling the autophagic flux, independently of its enzymatic activity and through regulation of Pi(4,5)P2 levels on lysosomes.****To test this hypothesis we we will characterize the PTEN- PLC signaling pathway (aim 1) and we will characterize the role of this pathway on Pi(4,5)P2 homeostasis and autophagy (aim 2).***

自噬，即自我吞噬的过程，从酵母到哺乳动物，在进化上都是保守的。该过程导致细胞器、蛋白质或核酸等胞质成分的溶酶体降解。自噬始于双膜囊的形成，该双膜囊扩张并形成可吞噬胞质成分的自噬体。然后自噬体与溶酶体融合，释放消化酶以促进货物降解。自噬调节亚细胞区室的动态重塑，并参与胚胎发生、饥饿反应、抗肿瘤发生和抗衰老等多种生理过程的控制。因此，了解这一重要过程对于基础研究和生物医学研究至关重要。****我们对控制磷酸肌醇循环的候选基因进行了细胞果蝇筛选，发现肌醇 5-磷酸酶 dOCRL 控制 PI(4,5)P2 稳态（Ben El Kadhi 等人，Current Biology 2011）。大部分 PI(4,5)P2 集中在质膜上，它参与几乎所有涉及细胞表面的事件。我们证明 dOCRL 与内体和溶酶体相关，并且它使溶酶体上的 PI(4,5)P2 去磷酸化，以将这种磷酸肌醇限制在质膜上。当 dOCRL 被 RNAi 敲除时，细胞在巨大溶酶体表面异常积累 PI(4,5)P2。此外，我们最近报道，PTEN 激活通过激活内膜上的磷脂酶 C (PLC) 来促进其自身酶产物 PI(4,5)P2 的水解。我们证明该功能可以挽救 dOCRL 损失（Ben El Kadhi 等人，准备中）。****在我们未发表的观察中，我们表明 dOCRL 的耗尽会导致自噬流缺陷。溶酶体不能再与自噬体融合。重要的是，我们发现激活 PTEN 可以恢复 dOCRL 耗尽细胞中的自噬流。我们还发现该功能独立于 PTEN 酶活性，但受到包含其两个非酶保守结构域的最小嵌合体的支持。此外，我们发现 PTEN 耗尽会促进 Pi(4,5)P2 在内膜上的积累。 ***除了通过抑制 PI3K/Akt/mTORC1 通路在自噬激活中发挥经典作用外，我们的结果表明 PTEN 在控制自噬通量方面具有新的作用，与其酶活性无关，并且通过调节溶酶体上的 Pi(4,5)P2 水平。****为了检验这一假设，我们将表征 PTEN-PLC 信号通路（目标 1）和 我们将描述该通路对 Pi(4,5)P2 稳态和自噬的作用（目标 2）。***