PI4KIIα in Late Stage Autophagy

后期自噬中的 PI4KIIα

• 批准号: 9216681
• 负责人: JOSEPH P ALBANESI
• 金额: $ 38.45万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9216681
• 关键词: 1-Phosphatidylinositol 4-Kinase; Adaptor Signaling Protein; Address; Affect; Autophagocytosis; Autophagosome; Binding; Biochemical; Biological; Biosensor; Cell physiology; Cells; Cholesterol; Complex; Data; Degradation Pathway; Detection; Disease; Energy Transfer; Ensure; Event; Family; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; GTP Binding; Gatekeeping; Golgi Apparatus; Grant; Growth; In Situ; Life Cycle Stages; Lipids; Lysosomes; Maps; Mediating; Membrane; Membrane Fusion; Modeling; Mutate; Organelles; Pathway interactions; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Protein Kinase; Proteins; RNA Interference; Recruitment Activity; Recycling; Regulation; Reporting; Research; Role; SERPINA4 gene; Scaffolding Protein; Source; Spectrum Analysis; Starvation; Structure; Surface; System; Testing; Vesicle; arm; base; body system; fluorescence lifetime imaging; human disease; late endosome; member; microvesicles; nanoimaging; novel; nutrient deprivation; overexpression; palmitoylation; phosphatidylinositol 4-phosphate; protein protein interaction; trafficking; trans-Golgi Network

PROJECT SUMMARY Autophagy is an essential homeostatic process. Under stressful conditions, such as nutrient deprivation, autophagy is markedly up-regulated to provide a source of raw materials to ensure survival and to dispose of damaged organelles. Autophagosomes are generated by elongation and closure of phagophores, and autophagosomes fuse with lysosomes to effect content degradation and recycling. Phosphoinositide lipids have been implicated in every stage of the autophagic pathway. However, to date, emphasis has been placed on the function of PI3P in phagophore elongation and closure. We found that PI4P is required for autophagosome fusion with lysosomes (A:L fusion), a culminating event at the end of the autophagy road. Furthermore, PI4K2A (PI4KIIα) is uniquely responsible for generating the PI4P on autophagosomes and it acts downstream of GABARAPs, Atg8 family autophagy proteins that are much less well understood than the LC3 Atg8 family. PI4K2A binds GABARAPs and associates with autophagosomes in a GABARAP- and palmitoylation-dependent manner. It is activated by starvation and translocates from the perinuclear Golgi region to autophagosomes. PI4K2A or GABARAP depletion blocks autophagy flux and A:L fusion, resulting in the formation of abnormally large autophagosomes. This block can be rescued by overexpressing PI4K2A or increasing intracellular PI4P by “PI4P shuttling.” We hypothesize that GABARAP promotes PI4K2A membrane trafficking to autophagosomes to generate PI4P in the GABARAP/PI4K2A interactome to activate the A:L fusion machinery. This hypothesis will be tested as follows: Aim I. Determine how GABARAPs target PI4K2A to autophagosomes. We will analyze PI4P dynamics and the temporal sequence of PI4K2A recruitment to autophagosomes. We will determine if GABARAP acts as a trafficking modulator to promote PI4K2A recruitment, and identify/disrupt the binding motifs to establish functionality of the interaction. Aim 2. Determine how PI4K2A and PI4P promote A:L fusion. We will focus on the role of PI4K2A in the recruitment and activation of Rab7, the master regulator of the A:L priming, tethering and fusion. We will test the hypothesis that Rab7 is targeted to autophagosomes by direct binding to PI4K2A or PI4P and/or by PI4P-mediated recruitment of the multivalent scaffolding protein, PLEKHM1. Mechanisms whereby PI4K2A/PI4P control the activity state of Rab7 will be examined. Aim 3. Determine how PI4K2A is regulated during autophagy. The effects of autophagy-dependent changes in PI4K2A phosphorylation and palmitoylation on targeting and kinase activity will be examined. A combination of biochemical, cell biological, and state-of-the-art fluorescence-based nanoimaging approaches will be employed to address these questions. This multi-PI grant will provide a comprehensive picture of the function and regulation of PI4K2A in its hitherto unexplored role in autophagy. In doing so, it will establish a novel paradigm for the regulation of autophagy and will address pressing problems in many human diseases associated with defective autophagy.

项目概要 自噬是在营养匮乏等应激条件下的重要稳态过程。 自噬显着上调，以提供确保生存和处理的原材料来源 受损的细胞器是通过吞噬泡的伸长和闭合产生的。 自噬体与溶酶体融合，影响磷酸肌醇脂质的降解和回收。 与自噬途径的各个阶段有关，但是迄今为止，重点已得到重视。 关于 PI3P 在吞噬细胞伸长和闭合中的功能，我们发现 PI4P 是吞噬细胞伸长和闭合所必需的。 自噬体与溶酶体融合（A:L 融合），是自噬之路终点的最终事件。 此外，PI4K2A (PI4KIIα) 独特地负责在自噬体上生成 PI4P，并且其作用 GABARAP 下游，Atg8 家族自噬蛋白，与 LC3 相比，人们对自噬蛋白的了解要少得多 Atg8 家族结合 GABARAP 并与 GABARAP 和自噬体结合。 它通过饥饿激活并从核周高尔基体转移。 PI4K2A 或 GABARAP 缺失会阻碍自噬通量和 A:L 融合，从而导致 异常大的自噬体的形成可以通过过度表达 PI4K2A 或 通过“PI4P 穿梭”增加细胞内 PI4P。我们发现 GABARAP 促进 PI4K2A 膜 转运至自噬体，在 GABARAP/PI4K2A 相互作用组中生成 PI4P，从而激活 A:L 该假设将按如下方式进行检验： 目标 I. 确定 GABARAP 如何靶向 PI4K2A 我们将分析 PI4P 动力学和 PI4K2A 招募的时间序列。 我们将确定 GABARAP 是否充当促进 PI4K2A 的运输调节剂。 招募，并识别/破坏结合基序以建立相互作用的功能。 PI4K2A 和 PI4P 如何促进 A:L 融合 我们将重点关注 PI4K2A 在招募和融合中的作用。 Rab7 的激活，A:L 启动、束缚和融合的主调节器我们将检验该假设。 Rab7 通过直接结合 PI4K2A 或 PI4P 和/或通过 PI4P 介导靶向自噬体 多价支架蛋白 PLEKHM1 的募集 PI4K2A/PI4P 控制的机制。 将检查 Rab7 的活性状态。 目标 3. 确定 PI4K2A 在自噬过程中如何受到调节。 自噬依赖性 PI4K2A 磷酸化和棕榈酰化变化对靶向和 将结合生物化学、细胞生物学和最先进的技术来检查激酶活性。 这项多 PI 资助将采用基于荧光的纳米成像方法来解决这些问题。 将全面介绍 PI4K2A 的功能和调节，以及其迄今为止尚未探索的作用 在此过程中，它将建立一个新的自噬调节范式，并将解决这一问题。 许多人类疾病中的紧迫问题与自噬缺陷有关。