Defining the role of ubiquitination in the regulation of lipid droplets

项目概要/摘要 为了开发新的治疗策略，以对抗日益流行的代谢性疾病（例如， 肥胖症、糖尿病和肝脂肪变性）的分子机制的深入了解， 脂肪的储存和流动是至关重要的。在细胞中，脂肪以三酰甘油的形式储存在脂滴（LD）中， 由磷脂包围的中性脂质核心组成的内质网衍生的细胞器 用嵌入的蛋白质装饰的单层。尽管LD蛋白质组在控制 LD功能，参与调节其活动和水平的机制知之甚少。 与泛素的翻译后缀合，其通过涉及E1、E2和E3的酶级联反应发生。 E3酶，几乎调节每一个细胞过程。我们的初步研究发现 调节LD泛素化途径，涉及UBXD 8介导的p97/VCP募集和调节LD 脂肪分解中的限速酶ATGL催化的周转。这些初步数据表明， 该通路在LD生物学中的功能重要性，并为进一步的机制研究提供基础。 本申请中描述的研究旨在检验UBXD 8作为传感器的假设 对于脂肪酸，其通过靶向代谢信号所需的酶来稳定LD， 用于Ub依赖性沉默的脂解。具体而言，本补助金中建议的研究将阐明 UBXD 8介导的ATGL功能抑制的机制（目的1），将构建一个全面的地图， LD相关的泛素化网络（目标2），并将确定LD泛素化的目标和他们的调控， 细胞的代谢状态（目标3）。 我在细胞生物学和生物化学方法的应用方面有丰富的经验， 了解泛素系统在细胞蛋白质质量和数量控制的各个方面的作用。 作为一名博士后学者，我已经扩大了我的研究训练，包括系统级战略， 定量蛋白质组学方法和LD功能测定。在K99阶段， 我将与我的导师罗恩Kopito博士和罗伯特Farese Jr.博士密切合作，公认的专家， 泛素生物学和脂质代谢领域，进行拟议的研究，并实施我的 研究和职业规划。在他们的指导下，我将受益于斯坦福大学的综合环境 大学和格莱斯顿心血管疾病研究所，共同提供了一个丰富的环境， 有机会进行实验培训，智力增长，合作，网络，职业生涯 发展和创新研究。我的短期目标是获得一个独立的职位 调查员，并在R00期间的奖励，我将制定一个强大的和原始的研究计划 重点是泛素对LD在健康和疾病中的功能的贡献。我的长期目标是 作为一个科学领导者和导师，以促进开拓性的研究，推进基本的 了解调节脂质代谢的分子机制，并最终导致 成功开发和实施治疗代谢性疾病的有效策略。

Project Summary / Abstract In order to develop novel therapeutic strategies to combat the increasing epidemic of metabolic diseases (eg. obesity, diabetes, and hepatic steatosis) an in-depth understanding of the molecular mechanisms underlying the storage and mobilization of fat is critical. In cells, fat is stored as triacylglycerol in lipid droplets (LDs), an endoplasmic reticulum-derived organelle composed of a neutral lipid core encircled by a phospholipid monolayer decorated with embedded proteins. Despite the clear importance of the LD proteome in controlling LD function, the mechanisms involved in regulating their activities and levels are poorly understood. Posttranslational conjugation with ubiquitin, which occurs via an enzymatic cascade involving an E1, E2, and E3 enzyme, regulates virtually every cellular process. Our preliminary findings identify a metabolically regulated LD ubiquitination pathway that involves UBXD8-mediated recruitment of p97/VCP and regulation LD turnover catalyzed by ATGL, the rate-limiting enzyme in lipolysis. These preliminary data establish the functional importance of this pathway in LD biology and provide a foundation for further mechanistic studies. The studies described in this application are designed to test the hypothesis that UBXD8 functions as a sensor for fatty acids that stabilizes LDs in response to metabolic signals by targeting the enzymes required for lipolysis for Ub-dependent silencing. Specifically, the studies proposed in this grant will elucidate the mechanism of UBXD8-mediated inhibition of ATGL function (Aim 1), will construct a comprehensive map of the LD-associated ubiquitination networks (Aim 2), and will identify LD ubiquitination targets and their regulation by the metabolic state of the cell (Aim 3). I have extensive experience in the application of cell biology and biochemistry approaches to understand the role of the ubiquitin system in various aspects of cellular protein quality and quantity control. As a postdoctoral scholar I have expanded my research training to include systems-level strategies, quantitative proteomics methodologies, and LD functional assays. During the mentored K99 phase of this award I will work closely with my mentors Dr. Ron Kopito and Dr. Robert Farese Jr., recognized experts in the fields of ubiquitin biology and lipid metabolism, to perform the proposed research and to implement my research and career plan. Under their mentorship I will benefit from the combined environments of Stanford University and the Gladstone Institute of Cardiovascular Disease, which together provide an environment rich with opportunities for experimental training, intellectual growth, collaborations, networking, career development, and innovative research. My short-term goal is to obtain a position as an independent investigator, and during the R00 period of the award I will develop a robust and original research program focused on the contribution of ubiquitin to LD function in health and disease. My long-term goal is to function as a scientific leader and mentor to promote pioneering research that advances the fundamental understanding of the molecular mechanisms regulating lipid metabolism and that ultimately leads to the successful development and implementation of efficacious strategies to treat metabolic diseases.