二酰甘油的稳态平衡及其对细胞内膜系统形态和功能的调控

Diacylglycerol (DAG) is both an intermediate lipid metabolite and a signaling molecule. Previously, we and others have shown that under starvation, yeast cells induce both autophagy and synthesis of triacylglycerol (TAG). Blocking TAG synthesis inhibits autophagy. However, the underlying mechanism remains elusive. In our preliminary work, we found, through systematic manipulation of metabolic pathways and careful characterization of cellular responses, that the main cause of autophagy inhibition and endomembrane system perturbation is the accumulation of DAG instead of phasphatidic acid (PA) or other upstream metabolites. Under such condition, elevation of DAG level in the endoplasmic reticulum (ER) alters the morphology of the ER and the balance of intracellular vesicular transports, through a mechanism independent of protein kinase C (PKC). By crossing to a knockout library, we performed a genome wide screen looking for mutants that can reverse the phenotypes of TAG synthesis inhibition. As expected, we found mutants that are impaired in phospholipid synthesis, and mutants functioning in vesicular trafficking. However, we also identified mutants primarily affecting sphingolipid synthesis. Based on these results, we propose to expand our research in the following two areas: one is to investigate how sphingolipid metabolism regulates DAG homeostasis, the other is to explore how DAG regulates the endomembrane system and autophagy. We expect that this work will bring new perspectives on the interplay between sphingolipid and phospholipid, and the physiological function of DAG.

二酰甘油既是代谢中间物，也是胞内信号分子。此前，我们和他人的工作均表明：饥饿的酵母细胞中细胞自噬和三酰甘油合成被上调，阻断三酰甘油合成可以抑制细胞自噬；但其机制众说纷纭。前期工作中，我们通过系统的生化通路操作和细胞生物学观察，确定此条件下自噬以及内质网高尔基体间囊泡运输被调控的关键在于二酰甘油的积累，而不是磷脂酸或更为上游的代谢物。此时，二酰甘油主要分布在内质网，以不依赖PKC的方式改变内质网形态和囊泡运输平衡。通过基因组突变体库杂交筛选，我们发现若干可在三酰甘油合成缺陷条件下恢复内质网、高尔基体状态和细胞自噬的突变体。除了磷脂代谢和囊泡运输相关蛋白，突变体还涉及参与鞘脂合成的酶。在此基础上，我们计划开展两方面研究：一是分析鞘脂代谢通路如何调控二酰甘油稳态，二是探索二酰甘油如何调控细胞内膜系统功能和细胞自噬。我们的工作有望为理解磷脂代谢与鞘脂代谢的联系、认识二酰甘油的生理功能提供新的视角。

生物膜的基本成分包括各种甘油脂、甾醇和鞘脂。组分的变化对膜的性质功能具有重大影响。本项目从酵母细胞自噬和细胞内膜结构角度研究甘油脂合成中间产物二酰甘油对生物膜功能的调控。项目研究发现，当酵母细胞用于贮存的中性脂三酰甘油合成被阻断时，二酰甘油含量提升且亚细胞分布从液泡向内质网转移。二酰甘油在内质网的积累造成内质网管网结构塌陷，内质网-高尔基体囊泡运输受到抑制，进而导致高尔基体解体、细胞自噬阻断在膜结构形成阶段。项目研究还发现二酰甘油对内膜系统的影响收到鞘脂调控。在鞘脂代谢突变体中，二酰甘油的总量基本不受影响，但亚细胞分布由内质网向质膜转移。通过对鞘脂代谢通路的梳理，实验证据提示调控二酰甘油状态的关键不是鞘脂总量，而是鞘脂特定分子结构的变化。项目研究过程中，课题组还对酵母脂质提取分析、细胞器光镜和电镜结构观察的方法工具进行了改进完善。本项工作为理解脂代谢紊乱的细胞生物学提供了新的视角，实验结果可以为多细胞生物相关研究提供借鉴。