The Mechanisms of Lipid Droplet Formation and Regulation

描述(申请人提供)：脂滴(LDS)是真核细胞中普遍存在的细胞器，组织和储存细胞脂，包括三酰甘油(TGS)，它提供能量和膜脂的来源。尽管最近在细胞生理学和疾病方面对LD生物学的了解取得了进展，但关于LDS的大多数基本关键问题仍然没有答案。在这项建议中，我们将重点放在最基本的问题上，即确定LD形成的机制。为了做到这一点，AIMS 1和2将使用我们开发的一种新系统，在该系统中，在哺乳动物细胞的零背景上诱导LD的形成。我们通过诱导酰基辅酶A：二酰甘油酰基转移酶(DGAT)1或DGAT2来实现这一点，它催化TG合成，位于LD形成过程的上游。通过在CEL中诱导LD的形成，我们将解决关键问题：TG合成如何与新形成的LD相协调？LDS在细胞中的什么位置形成？单层结合的LDS是如何从双层膜中形成的？具体地说，目标1侧重于在LD形成之前、期间和后期确定DGAT酶的定位和活性。目的2着重于确定LD在细胞中的形成位置，LD的形成与膜拓扑结构、管状(或弯曲的)内质网的关系，以及内质网中的TGs在LD形成过程中是如何组织的。最终目标集中在我们的发现上，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LDS的。因此，目标3将阐明Core靶向LDS的机制，并将识别通过这一机制获得LDS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的形成可能对LD过剩的疾病具有治疗意义，如肥胖、糖尿病和动脉粥样硬化，并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。 公共卫生相关性：脂肪过多的代谢性疾病，如肥胖、2型糖尿病和动脉粥样硬化，是非常普遍的疾病。这些疾病中多余的脂肪储存在细胞中的脂滴或脂滴中。令人惊讶的是，人们对细胞生物学的这一方面知之甚少。我们的研究重点是了解脂肪液滴如何在细胞中形成的基本生物学。我们的工作将有助于更好地了解脂肪过多的疾病，并可能导致新的治疗方法。

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. PUBLIC HEALTH RELEVANCE: Metabolic diseases of fat excess, such as obesity, type 2 diabetes, and atherosclerosis, are hugely prevalent disorders. The excess fat in these disorders is stored in cells in fat droplets or lipid droplets. Remarkably little is known about this aspect of cell biology. Our studies are focused on understanding the basic biology that underlies how fat droplets are formed in cells. Our work will lead to a greater understanding of diseases of fat excess and may lead to new therapies.