Mechanism of Lipid Droplet/ Mitochondria Contacts and Role of Perilipin 5 in Lipid Metabolism

脂滴/线粒体接触机制及 Perilipin 5 在脂质代谢中的作用

• 批准号: 10065326
• 负责人: Gregory Miner
• 金额: $ 6.53万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065326
• 关键词: Affinity Chromatography; Atherosclerosis; Binding; Biochemical; Biological Assay; Brown Fat; C-terminal; Cachexia; Cells; Cellular biology; Communities; Data; Development; Disease; Educational workshop; Equilibrium; Essential Fatty Acids; Family member; Fatty Acids; Fatty Liver; Goals; Heart; Human Cell Line; Image; Impairment; Lead; Length; Lipase; Lipid Mobilization; Lipids; Lipodystrophy; Lipolysis; Liver diseases; Mass Spectrum Analysis; Mediating; Membrane; Membrane Lipids; Mentors; Metabolic syndrome; Metabolism; Methods; Mission; Mitochondria; Morphology; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Organelles; Outcome; Outer Mitochondrial Membrane; Oxides; Pathway interactions; Phospholipids; Phosphorylation; Physiologic pulse; Physiological; Physiology; Play; Protein Family; Proteins; Proteomics; Public Health; Radioactive; Research; Research Training; Role; Site; Skeletal Muscle; Starvation; Techniques; Testing; Tissues; Training; Triglycerides; United States National Institutes of Health; Western Blotting; Work; base; career development; disability; fatty acid metabolism; fatty acid oxidation; human disease; improved; insight; light microscopy; lipid metabolism; lipid transport; monolayer; novel; novel therapeutics; nutrient deprivation; overexpression; perilipin; prevent; response; small hairpin RNA; sterol ester; trafficking

PROJECT SUMMARY Fatty acids (FAs) are essential for cellular energy storage and as the precursors for synthesis of many lipids. FAs are predominantly stored as triglycerides in organelles called lipid droplets (LDs). LDs form dynamic membrane contacts with other organelles including mitochondria (Mito), a site of FA oxidation. These contacts are proposed to facilitate the trafficking of lipids between organelles. Impaired FA trafficking and metabolism can lead to a variety of serious diseases. Lipodystrophy and cachexia are caused by deficient lipid storage, while the metabolic syndrome, type 2 diabetes, atherosclerosis and fatty liver disease involve excess lipid storage. Therefore, there is a critical need to identify the mechanism and physiological functions of LD- organelle contacts. Perilipin 5 (Plin5) is an LD protein that mediates LD-Mito contacts. The overall objectives are to (i) identify the mechanism by which Plin5 induces LD-Mito contacts (ii), to determine the role of these contacts in FA trafficking, and (iii) to evaluate the effect of Plin5 on lipid metabolism organelles. The central hypothesis is that Plin5 utilizes a unique C-terminal domain to induce LD-Mito contacts that promote β- oxidization during nutrient starvation by directing FA trafficking from LDs to Mito. The rationale for this project is that identifying the mechanism and physiological role of LD-Mito contacts will result in strategies to treat lipid storage/trafficking diseases. The central hypothesis will be tested by pursuing three specific aims. In Aim 1, affinity purification mass spectrometry of Plin5 constructs lacking the minimal LD-Mito contact domain will be performed to identify mitochondrial binding partners. Identified binding partners will then be assessed for a role in LD-Mito contact formation. In Aim 2, fluorescent and radioactive pulse-chase assays will be utilized to identify the physiological effect of Plin5 on FA storage, trafficking and metabolism. In Aim 3, multispectral imaging will be utilized to evaluate the effect of Plin5 on the morphology and contacts between 5 organelles involved in lipid metabolism. The expected outcomes are to define the mechanism by which Plin5 induces LD- Mito contacts and to demonstrate the physiological function of these contacts. These results will have important positive impact because they will establish a strong basis from which to further study dysregulated lipid trafficking and suggest a framework from which to develop novel therapies for lipid-associated diseases. To enable these research objectives, mentoring and technical/theoretical training will be provided by Dr. Cohen, Dr. Coleman, and Dr. Klett whom are experts in multispectral imaging and lipid trafficking/metabolism respectively. Career development will be facilitated by the Office of Postdoctoral Affairs through seminars and workshops. The proposed research and training will be conducted at UNC Chapel Hill, which is well known for its outstanding cell biology community, including extensive expertise in membrane trafficking and the development of novel techniques in light microscopy.

项目摘要 脂肪酸（FAs）是细胞能量储存和许多脂质合成的前体所必需的。 脂肪酸主要以甘油三酯的形式储存在称为脂滴（LD）的细胞器中。LD形式动态 膜与其他细胞器接触，包括线粒体（Mito），FA氧化的位点。这些接触 被提议促进脂质在细胞器之间的运输。FA运输和代谢受损 可导致多种严重疾病。脂肪营养不良和恶病质是由脂质储存不足引起的， 而代谢综合征、2型糖尿病、动脉粥样硬化和脂肪肝疾病涉及过量脂质 存储.因此，迫切需要确定LD的机制和生理功能。 细胞器接触。Perilipin 5（Plin 5）是介导LD-Mito接触的LD蛋白。总体目标 是（i）确定Plin 5诱导LD-Mito接触的机制（ii），以确定这些接触的作用。 接触FA运输，和（iii）评估Plin 5对脂质代谢细胞器的影响。中央 假设Plin 5利用独特的C-末端结构域诱导LD-Mito接触，其促进β- 氧化过程中营养饥饿的指导FA运输从LD水户。该项目的基本原理是 确定LD-Mito接触的机制和生理作用将导致治疗脂质的策略， 储存/贩运疾病。中心假设将通过追求三个具体目标来检验。在目标1中， 缺乏最小LD-Mito接触结构域的Plin 5构建体的亲和纯化质谱将被 用于鉴定线粒体结合伴侣。然后将评估已识别的绑定伙伴的作用 在LD-Mito接触形成。在目标2中，将利用荧光和放射性脉冲追踪分析， 鉴定Plin 5对FA储存、运输和代谢的生理作用。在目标3中， 将利用成像来评估Plin 5对5个细胞器之间的形态和接触的影响 参与脂质代谢。预期的结果是确定Plin 5诱导LD的机制。 美图接触，并展示这些接触的生理功能。这些结果将 重要的积极影响，因为它们将为进一步研究失调奠定坚实的基础 脂质运输，并提出了一个框架，从中开发新的治疗脂质相关疾病。 为了实现这些研究目标，指导和技术/理论培训将提供博士。 Cohen、科尔曼博士和Klett博士，他们是多光谱成像和脂质运输/代谢方面的专家 分别博士后事务办公室将通过研讨会和 工作坊.拟议的研究和培训将在查佩尔山进行，这是众所周知的， 其杰出的细胞生物学社区，包括在膜运输和 光学显微镜新技术的发展。