Mechanisms of Lipid Droplet Protein Targeting

脂滴蛋白靶向机制

• 批准号: 8080650
• 负责人: Tobias C Walther
• 金额: $ 41.4万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080650
• 关键词: Affect; Atherosclerosis; Biochemical; Biogenesis; Biological; Biological Models; Cell physiology; Cells; Cellular biology; Complement; Cytoplasm; Cytosol; Disease; Drosophila genus; Endoplasmic Reticulum; Enzymes; Fatty acid glycerol esters; Focus Groups; Genes; Goals; Health; Hepatitis C virus; Homeostasis; Human; In Vitro; Institutes; Integral Membrane Protein; Intervention; Laboratories; Lead; Letters; Lipid Bilayers; Lipids; Lipolysis; Mammalian Cell; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metabolic; Metabolic Diseases; Metabolic syndrome; Metabolism; Methods; Microscopy; Modeling; Morphology; Nature; Obesity; Organelles; Pathway interactions; Phase; Phenotype; Phospholipids; Process; Proteins; Proteome; Proteomics; RNA Interference; Recruitment Activity; Research; Research Subjects; Set protein; Side; Signal Transduction; Surface; Testing; Time; Triglycerides; Viral Proteins; Virus Diseases; Virus Replication; aqueous; base; cofactor; combat; in vivo; medical specialties; membrane biogenesis; monolayer; sterol ester; therapeutic development; treatment site

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.

描述（由申请人提供）：细胞代谢能量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 还含有膜生物发生所需的甾醇酯 (SE)。 LD的过度积累发生在某些疾病的进展过程中，包括肥胖、代谢综合征和动脉粥样硬化。 LD 也是丙型肝炎病毒复制所必需的，并且许多病毒蛋白与该细胞器特异性相互作用。尽管 LD 细胞对人类健康具有重要意义，但令人惊讶的是，人们对 LD 细胞的基本生物学知之甚少，尤其是在蛋白质靶向方面。由 TG 和 SE 组成的 LD 的疏水核心以磷脂单层为界，其中包含一组很大程度上未识别的蛋白质。 LD 的大多数功能，包括 TG 合成、TG 存储和能量动员，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）的并置。因此，该单层不适合容纳具有侧翼跨膜片段的球状结构域的典型跨膜蛋白。因此，将特定蛋白质靶向 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，我们必须首先确定高置信度的 LD 蛋白质组，这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实现。然后，我们将继续确定蛋白质如何从细胞质靶向 LD，重点关注一组模型蛋白质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，我们将使用公正的蛋白质组学方法来定义共享为这些模型定义的途径的蛋白质的范围。在第二项研究中，我们建议确定一组不同的蛋白质（包含预测多个跨膜结构域的序列）如何靶向 LD 或紧密相关的膜。对于该项目的每个部分，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守性。尽管这里提出的研究是基础性的，但我们对驱动脂滴蛋白靶向的基本细胞机制的确定将有助于开发对抗涉及脂滴的疾病的治疗策略，并推动对这些细胞器的细胞生物学的进一步研究。 公共卫生相关性：脂滴在能量储存、新陈代谢和体内平衡中发挥作用。它们由独特的磷脂单层包围，其中包含在这些过程中具有功能的特定蛋白质。我们在此建议建立 LD 蛋白质组，以确定这些蛋白质能够实现其专门靶向的特征，确定发生靶向所需的因素，并表征靶向如何参与调节 LD 的功能。我们的结果将定义一个未经研究的基本细胞过程，为探索推测的 LD 功能提供基础，并为治疗各种代谢和病毒疾病建立干预位点。