Defining the role of ubiquitination in the regulation of lipid droplets

项目摘要/摘要 为了开发新的治疗策略来对抗日益流行的代谢性疾病(例如， 肥胖、糖尿病和肝脏脂肪变性)深入了解其潜在的分子机制 脂肪的储存和动员是至关重要的。在细胞中，脂肪以三酰甘油的形式储存在脂滴(LDS)中，一种 内质网衍生的细胞器，由被磷脂包裹的中性脂核组成 用嵌入蛋白质装饰的单层。尽管LD蛋白质组在控制 对于LD的功能，调节其活动和水平的机制知之甚少。 翻译后与泛素的结合，这是通过涉及E1、E2和 E3酶，几乎调节每一个细胞过程。我们的初步发现确定了一种新陈代谢 参与UBXD8介导的p97/VCP募集的受控LD泛素化途径及其调控 由脂肪分解中的限速酶ATGL催化的周转。这些初步数据确立了 该途径在LD生物学中的功能重要性，并为进一步的机制研究提供了基础。 本申请中描述的研究旨在测试UBXD8作为传感器的假设 对于通过靶向所需的酶来稳定LDS以响应代谢信号的脂肪酸 Ub依赖沉默的脂解作用。具体地说，这笔拨款建议的研究将阐明 UBXD8介导的ATGL功能抑制的机制(目标1)，将构建一个全面的图谱 与LD相关的泛素化网络(AIM 2)，并将确定LD泛素化靶标及其调控 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，我已经将我的研究培训扩展到包括系统级战略， 定量蛋白质组学方法和LD功能分析。在此的指导K99阶段 我将与我的导师Ron Kopito博士和Robert Farese Jr.博士密切合作，他们是 泛素生物学和脂类代谢领域，以执行拟议的研究和实施我的 研究和职业规划。在他们的指导下，我将从斯坦福的综合环境中受益 大学和格莱斯顿心血管疾病研究所，共同提供了丰富的环境 提供实验性培训、智力增长、协作、网络、职业发展等机会 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，在获奖的R00期间，我将制定一个强有力的原创研究计划 重点阐述了泛素在健康和疾病中对LD功能的贡献。我的长期目标是发挥作用 作为科学领导者和导师，推动开创性研究，推动 了解调节脂类代谢的分子机制，并最终导致 成功制定和实施治疗代谢性疾病的有效战略。

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.