The Mechanisms of Lipid Droplet Formation and Regulation

描述（由申请人提供）：脂滴（LD）是真核细胞中普遍存在的细胞器，用于组织和储存细胞脂质，包括三酰甘油（TG），提供能量储存和膜脂来源。尽管最近在理解LD生物学在细胞生理学和疾病方面取得了进展，但关于LD的最基本的关键问题仍然没有答案。在这个建议中，我们专注于最基本的问题，确定潜在的LD形成的机制。为此，目标1和2将采用我们开发的新系统，其中在哺乳动物细胞中在空背景下诱导LD形成。我们通过诱导酰基辅酶A：二酰基甘油酰基转移酶（DGAT）1或DGAT 2来实现这一点，它们催化TG合成并位于LD形成过程的上游。通过诱导LD的形成，我们将解决关键问题：TG合成如何与新形成的LD协调？LD在细胞的什么地方形成？如何从双层膜形成单层结合的LD？具体而言，目标1侧重于确定在LD形成之前、期间和后期阶段DGAT酶的定位和活性。目的2侧重于确定LD形成发生在细胞中，LD形成如何与膜拓扑结构，管状（或弯曲）ER，以及在LD形成期间ER中的TG如何组织。最终的目标集中在我们的发现，即特定的蛋白质，如丙型肝炎核心蛋白，显然是针对新形成的LD。因此，目的3将阐明核心靶向LD的机制，并将鉴定通过该机制获得LD的其他内源性细胞蛋白。我们的研究将提供有关LDs和细胞脂质代谢的基础知识。此外，更好地了解LD形成可能对LD过量的疾病，如肥胖症，糖尿病和动脉粥样硬化具有治疗意义，并且可能在工程植物和微生物中具有实际应用以增加石油产量。

DESCRIPTION (provided by applicant): Lipid droplets (LDs) are ubiquitous organelles in eukaryotic cells that organize and store cellular lipids, including triacylglycerols (TGs), which provide stores of energy and sources of membrane lipids. Despite recent advances in understanding LD biology in cellular physiology and disease, most fundamental key questions about LDs remain unanswered. In this proposal, we focus on the most basic question of determining the mechanisms underlying LD formation. To do so, Aims 1 and 2 will employ a novel system we developed, in which LD formation is induced on a null background in mammalian cells. We accomplish this by inducing acyl CoA: diacylglycerol acyltransferase (DGAT) 1 or DGAT2, which catalyze TG synthesis and lie just upstream to the process of LD formation. By inducing LD formation in cels, we will address key questions: How is TG synthesis coordinated with newly forming LDs? Where in the cell do LDs form? How do monolayer-bound LDs form from bilayer membranes? Specifically, Aim 1 focuses on determining the localization and activity of DGAT enzymes before, during, and at later stages of LD formation. Aim 2 focuses on determining where LD formation occurs in the cell, how LD formation relates to membrane topology, tubular (or curved) ER, and how TGs in the ER are organized during LD formation. The final aim focuses on our findings that specific proteins, such as the hepatitis C Core protein, are apparently targeted to newly forming LDs. Aim 3 therefore will elucidate the mechanism that underlies the targeting of Core to LDs and will identify other endogenous cellular proteins that gain access to LDs through this mechanism. Our studies will provide fundamental knowledge about LDs and cellular lipid metabolism. Additionally, a better understanding of LD formation may have therapeutic implications for diseases of LD excess, such as obesity, diabetes, and atherosclerosis, and may have practical applications in engineering plants and microorganisms for increased oil production.