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

项目总结 脂肪酸(FAs)是细胞能量储存所必需的，也是合成多种脂质的前体。 Fas主要以甘油三酯的形式储存在被称为脂滴(LDS)的细胞器中。LDS表单动态 膜与其他细胞器接触，包括线粒体(MITO)，这是FA氧化的部位。这些联系人 建议用来促进细胞器之间的脂类运输。FA转运和代谢受损 会导致多种严重疾病。脂肪营养不良和恶病质是由脂肪储存不足引起的， 而代谢综合征、2型糖尿病、动脉粥样硬化和脂肪肝则涉及过多的脂肪 储藏室。因此，迫切需要确定LD的机制和生理功能。 细胞器接触。Perilipin 5(Plin5)是一种介导LD-Mito接触的LD蛋白。总体目标 目的是(I)确定Plin5诱导LD-Mito接触的机制(Ii)，以确定这些机制的作用 (3)评价Plin5对脂类代谢细胞器的影响。中环 假设Plin5利用一个独特的C-末端结构域诱导LD-MITO接触，从而促进β- 营养饥饿期间的氧化通过引导FA从LDS运输到Mito。这个项目的基本原理是 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。核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Plin5结构的亲和纯化质谱学将是 用来识别线粒体结合伙伴。然后将评估确定的绑定合作伙伴的角色 在LD-Mito接触队形中。在目标2中，将利用荧光和放射性脉冲追逐分析来 明确Plin5在脂肪酸储存、运输和代谢中的生理作用。目标3，多光谱 将利用成像来评估Plin5对5个细胞器的形态和接触的影响 参与脂类代谢的。预期的结果是确定Plin5诱导LD的机制。 Mito触点，并演示这些触点的生理功能。这些结果将会有 重要的积极影响，因为它们将为进一步研究监管失调奠定坚实的基础 此外，还提出了一个框架，用以开发治疗与脂质相关的疾病的新疗法。 为了实现这些研究目标，博士将提供指导和技术/理论培训。 科恩、科尔曼博士和克莱特博士，他们是多光谱成像和脂肪运输/代谢方面的专家 分别进行了分析。博士后事务办公室将通过研讨会和 研讨会。拟议的研究和培训将在北卡罗来纳大学教堂山进行，那里以 其杰出的细胞生物学社区，包括在膜贩运和 光学显微镜新技术的发展。

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.