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

自噬是自噬的过程，从酵母到哺乳动物，在进化上都是保守的。这一过程导致细胞器、蛋白质或核酸等胞液成分的溶酶体降解。自噬始于双膜囊的形成，该双膜囊扩张并形成能吞噬胞液成分的自噬小体。然后，自噬小体与释放消化酶的溶酶体融合，以促进货物的降解。自噬调节亚细胞室的动态重塑，参与控制胚胎发生、饥饿反应、抗肿瘤和抗衰老等生理过程。因此，对这一重要过程的了解对于基础和生物医学研究都是至关重要的。*我们在纤维果蝇中对控制肌醇肌醇循环的候选基因进行了筛选，我们发现肌醇5-磷酸酶dOCRL控制PI(4，5)P2动态平衡(Ben El Kadhi等人，当代生物学，2011)。PI(4，5)P2的大部分集中在质膜上，在那里它参与了几乎所有涉及细胞表面的事件。我们证明dOCRL与内小体和溶酶体有关，并且它使溶酶体上的PI(4，5)P2去磷酸化，从而限制质膜上的磷脂酰肌醇。当dOCRL被RNAi敲除时，细胞在巨型溶酶体表面异常积聚PI(4，5)P2。此外，我们最近报道，PTEN的激活通过激活内膜上的磷脂酶C(PLC)酶来促进其自身的酶产物PI(4，5)P2的水解。我们证明了这个功能可以挽救dOCRL的丢失(Ben El Kadhi等人，在PREP中)。*在我们未发表的观察中，我们表明dOCRL的耗尽导致自噬通量的缺陷。溶酶体不能再与自噬小体融合。重要的是，我们发现激活PTEN可以恢复dOCRL耗竭细胞的自噬通量。我们还发现，这一功能不依赖于PTEN的酶活性，但由包含其两个非酶保守结构域的最小嵌合体支持。此外，我们还发现PTEN的缺失促进了PI(4，5)P2在细胞内膜上的积累。*除了它通过抑制PI3K/Akt/mTORC1途径在自噬激活中的典型作用外，我们的结果还表明，PTEN在控制自噬通量方面发挥了一个新的作用，不依赖于它的酶活性，并通过调节溶酶体上的PI(4，5)P2水平。*为了验证这一假说，我们将描述PTEN-PLC信号通路(目标1)，并描述该通路在PI(4，5)P2动态平衡和自噬中的作用(目标2)。

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).***