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，包括三酰基甘油（tg），它提供能量储存和膜脂的来源。尽管最近在细胞生理学和疾病方面对LD生物学的理解取得了进展，但关于LD的大多数基本关键问题仍未得到解答。在本建议中，我们将重点放在确定LD形成机制的最基本问题上。为此，Aims 1和Aims 2将采用我们开发的一种新系统，在哺乳动物细胞中在零背景下诱导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DGAT2来实现这一目标，它们催化TG合成，位于LD形成过程的上游。通过在细胞中诱导LD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？单层结合的ld是如何从双层膜形成的？具体来说，Aim 1侧重于确定LD形成前、期间和后期DGAT酶的定位和活性。目的2侧重于确定LD在细胞中的形成位置，LD的形成与膜拓扑、管状（或弯曲）内质网的关系，以及LD形成过程中内质网中的tg是如何组织的。最后的目标集中在我们的发现，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，Aim 3将阐明Core靶向ld的机制，并将确定通过该机制获得ld的其他内源性细胞蛋白。我们的研究将提供ld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过量疾病（如肥胖、糖尿病和动脉粥样硬化）具有治疗意义，并可能在工程植物和微生物中有实际应用，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.