Mechanisms of Lipid Droplet Protein Targeting

描述（由申请人提供）：细胞代谢能以中性脂质的形式储存，特别是三酰甘油（tg），其包装在细胞质脂滴（ld）中。ld还含有甾醇酯（SEs），这是膜生物发生所必需的。在某些疾病的发展过程中，包括肥胖、代谢综合征和动脉粥样硬化，ldl会过度积累。lld也是丙型肝炎病毒复制所必需的，许多病毒蛋白特异性地与这种细胞器相互作用。尽管它们对人类健康具有重要意义，但令人惊讶的是，人们对基本的LD细胞生物学知之甚少，特别是在蛋白质靶向方面。由TGs和SEs组成的ld的疏水核心由磷脂单层包围，其中包含一组大部分未识别的蛋白质。ld的大部分功能，包括TG合成、TG储存和能量动员，都是由这些表面蛋白执行和调节的。在细胞器中，LD是独特的，因为它们的表面是疏水相（LD核心）和水相（细胞质）的结合。因此，这种单层结构不能容纳典型的跨膜蛋白，其球形结构域位于跨膜片段的两侧。因此，特定蛋白靶向ld必须涉及独特的机制，这是本文提出的研究主题。为了阐明这些机制，我们必须首先确定一个高可信度的LD蛋白质组，我们可以通过我们最先进的基于定量质谱的蛋白质组学方法来完成。然后，我们将继续确定蛋白质如何靶向细胞质中的ld，重点关注一组模型蛋白，并应用我们在实验室建立的各种细胞生物学和生化方法。随后，我们将使用我们的无偏蛋白质组学方法来定义共享这些模型定义的途径的蛋白质范围。在第二项研究中，我们建议确定一组包含预测多个跨膜结构域序列的独特蛋白质如何靶向ld或密切相关的膜。对于项目的每个部分，我们将测试LD蛋白靶向的功能后果以及我们发现的机制的进化保护。虽然这里提出的研究是基础的，但我们对驱动脂滴蛋白靶向的基本细胞机制的确定将促进对抗涉及脂滴的疾病的治疗策略的发展，并推动对这些细胞器的细胞生物学的进一步研究。

DESCRIPTION (provided by applicant): Cellular metabolic energy is stored in the form of neutral lipids, particularly triacylglycerols (TGs), which are packaged in cytoplasmic lipid droplets (LDs). LDs also contain sterol esters (SEs), which are required for membrane biogenesis. Excessive accumulation of LDs occurs during the progression of certain diseases, including obesity, metabolic syndrome and atherosclerosis. LDs are also required for hepatitis C virus replication, and a number of viral proteins specifically interact with this organelle. Despite their significance to human health, surprisingly little is known about basic LD cell biology, especially in terms of protein targeting. The hydrophobic core of LDs consisting of TGs and SEs is bounded by a phospholipid monolayer, which harbors a set of largely-unidentified proteins. Most functions of LDs, including TG synthesis, TG storage, and energy mobilization, are executed and regulated by these surface proteins. Among organelles, LDs are unique because their surface is an apposition of a hydrophobic phase (the LD core) and an aqueous phase (the cytoplasm). This monolayer is thus not configured to accommodate typical transmembrane proteins with globular domains flanking transmembrane segments. Therefore, the targeting of particular proteins to LDs must involve unique mechanisms, which are the subject of the research proposed here. To elucidate these mechanisms, we must first determine a high-confidence LD proteome, which we can accomplish via our state-of-the-art quantitative mass spectrometry-based proteomics methods. We will then proceed to determine how proteins are targeted to LDs from the cytoplasm, focusing on a set of model proteins and applying a variety of cell biological and biochemical methods we have established in our laboratory. We will subsequently use our unbiased proteomics approach to define the scope of proteins that share the pathways defined for these models. In a second line of research, we propose to determine how a distinct set of proteins that contain sequences predicting multiple trans-membrane domains are targeted to LDs or closely- associated membranes. For each part of the project, we will test the functional consequences of LD protein targeting as well as evolutionary conservation of the mechanisms we discover. Although the research proposed here is basic, our determination of the fundamental cellular mechanism that drives lipid droplet protein targeting will facilitate the development of therapeutic strategies to combat diseases that involve lipid droplets, as well as propel further research into the cell biology of these organelles. PUBLIC HEALTH RELEVANCE: Lipid Droplets function in energy storage, metabolism and homeostasis. They are bounded by a unique phospholipid monolayer that contains specific proteins with functions in these processes. We propose here to establish the LD proteome, to determine the features of these proteins that enable their specialized targeting, to identify factors required for targeting to occur, and to characterize how targeting participates in regulating the functions of LDs. Our results will define an unstudied fundamental cellular process, provide a basis for exploring speculated LD functions, and establish an intervention site for the treatment of various metabolic and viral diseases.