The role of phosphatidylinositol 3-phosphate (PI3P) in the induction of autophagy

磷脂酰肌醇 3-磷酸 (PI3P) 在诱导自噬中的作用

• 批准号: BB/G016607/1
• 金额: $ 9.48万
• 依托单位: Babraham Institute
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG016607%2F1
• 关键词: role; phosphatidylinositol; phosphate; PI3P; induction

Autophagy is an important cellular response to nutrient limitation because it allows the cell to survive for a finite length of time by self digestion of its own components to generate nutrients. Autophagy is also an important contributor to quality control mechanisms during normal growth and development, and as such it has been shown to be critical for maintaining proper life span. Finally, several cancer models have been found to either up- or down-regulate autophagy, indicating that autophagy may also be involved in tumour progression (Genes Dev 21: 2861-73, 2007). The induction of autophagy represents a critical commitment step and is regulated tightly. Among the upstream regulators are mechanisms (as yet unknown) for nutrient sensing and proteins involved in PI 3-kinase signalling including the mammalian target of rapamycin kinase. How these regulators co-ordinate to provide the autophagy signal is currently under intense investigation. What is more clear from recent work is that the signal ultimately results in formation of PI3P, and it is this lipid that allows autophagy to proceed (Autophagy 4: 952-4, 2008). However, the exact function of PI3P in the induction step was unknown until recently. By following the dynamics of several PI3P-binding proteins during amino acid (AA) starvation in live cells we have provided some clues as to the function of PI3P in autophagy induction (J Cell Biol 182: 685-701, 2008). We found that PI3P starts to accumulate soon after AA starvation in novel membrane compartments that we termed omegasomes. These omegasomes are in dynamic equilibrium with the endoplasmic reticulum, and they constitute sites of autophagosome biogenesis. Therefore, our recent data provide an explanation of the role of PI3P in early autophagy. The aim of this studentship will be to further explore the role of PI3P in autophagy induction. The majority of the work will involve methods for the visualization of PI3P in omegasomes, during the earliest stages of the starvation response. We propose to do this work in collaboration with Innova Biosciences, a company that has broad expertise in generating fluorescent bio-conjugates. These bio-conjugates (in the form of antibodies or lipid-binding domains) will be essential for a successful outcome. In one part of the project the student will generate fluorescent probes specific for the omegasome-restricted pool of PI3P to evaluate starvation responses in a broad number of cell systems and in tissues. To achieve this we will generate recombinant versions of our ER-FYVE probes tagged to GST and we will use the Innova technology to conjugate them to fluorescent reporters such as FITC or RITC. In a parallel approach, we will generate antibodies against DFCP1, the only endogenous protein known so far to reside in omegasomes, and we will again conjugate these antibodies with fluorescent reporters in order to generate useful probes. Once these probes are at hand, we will use them to examine autophagy-specific PI3P accumulation in cells during starvation and in (a) tissues from control and starved animals and (b) from normal vs tumour tissue samples. This type of experiment will provide important information on the contribution of autophagy to pathological or physiological states. A second related aim will be the identification of the PI3P phospholipid phosphatase responsible for terminating the induction signal during autophagy. In animal cells there are 14 members of the myotubularin-related proteins (MTMRs, Trends Cell Biol 16: 403-12, 2006) and we expect that one or more will be responsible for de-phosphorylating PI3P once it has fulfilled its function. To identify these enzymes we will use siRNA against all members of the family and follow omegasome dynamics. We expect that knock-down of the relevant gene will lead to an increase in PI3P in omegasomes, and it may even be sufficient to increase basal autophagy. Assays for these outcomes are established in my group.

自噬是一种重要的细胞对营养限制的反应，因为它允许细胞通过自我消化其自身组分以产生营养物而存活有限的时间长度。自噬也是正常生长和发育过程中质量控制机制的重要贡献者，因此它已被证明对维持适当的寿命至关重要。最后，已经发现几种癌症模型上调或下调自噬，表明自噬也可能参与肿瘤进展（Genes Dev 21：2861-73，2007）。自噬的诱导是一个关键的承诺步骤，并受到严格的监管。在上游调节器中，有营养传感机制（目前尚不清楚）和PI 3-激酶信号传导中涉及的蛋白质，包括哺乳动物雷帕霉素激酶靶标。这些调节器如何协调提供自噬信号目前正在进行深入研究。从最近的工作中更清楚的是，信号最终导致PI 3 P的形成，并且正是这种脂质允许自噬进行（Autophagy 4：952-4，2008）。然而，直到最近，PI 3 P在诱导步骤中的确切功能才为人所知。通过跟踪活细胞中氨基酸（AA）饥饿期间几种PI 3 P结合蛋白的动力学，我们提供了关于PI 3 P在自噬诱导中的功能的一些线索（J Cell Biol 182：685-701，2008）。我们发现，PI 3 P开始积累后不久，AA饥饿在新的膜隔室，我们称之为omegasomes。这些omegasome与内质网处于动态平衡，它们构成自噬体生物发生的位点。因此，我们最近的数据提供了PI 3 P在早期自噬中的作用的解释。本研究的目的是进一步探索PI 3 P在自噬诱导中的作用。大部分工作将涉及在饥饿反应的最早阶段，在omegasomes中可视化PI 3 P的方法。我们建议与Innova Biosciences合作开展这项工作，Innova Biosciences是一家在生成荧光生物缀合物方面拥有广泛专业知识的公司。这些生物缀合物（以抗体或脂质结合结构域的形式）对于成功的结果至关重要。在该项目的一部分，学生将产生特异性的ω体限制池的PI 3 P的荧光探针，以评估饥饿反应在广泛的细胞系统和组织。为了实现这一目标，我们将产生标记GST的ER-FYVE探针的重组版本，我们将使用Innova技术将其与荧光报告分子（如FITC或RITC）偶联。在平行方法中，我们将产生针对DFCP 1的抗体，DFCP 1是迄今为止已知存在于omegasome中的唯一内源性蛋白质，并且我们将再次将这些抗体与荧光报告分子缀合以产生有用的探针。一旦这些探针在手，我们将使用它们来检查自噬特异性PI 3 P在饥饿期间细胞中的积累，以及在（a）对照和饥饿动物的组织和（B）正常与肿瘤组织样品中的积累。这种类型的实验将提供关于自噬对病理或生理状态的贡献的重要信息。第二个相关的目标将是鉴定负责终止自噬过程中诱导信号的PI 3 P磷脂磷酸酶。在动物细胞中，存在14种肌微管蛋白相关蛋白的成员（MTMR，Trends Cell Biol 16：403-12，2006），并且我们预期一旦PI 3 β完成其功能，一种或多种将负责去磷酸化PI 3 β。为了鉴定这些酶，我们将使用针对该家族所有成员的siRNA并遵循omegasome动力学。我们预计相关基因的敲低将导致omegasomes中PI 3 P的增加，甚至可能足以增加基础自噬。在我的小组中建立了这些结果的测定。