Defining the role of ubiquitination in the regulation of lipid droplet function

描述(申请人提供)：为了开发新的治疗策略来对抗日益流行的代谢性疾病(如肥胖症、糖尿病和肝脏脂肪变性)，深入了解脂肪储存和动员的分子机制是至关重要的。在细胞中，脂肪以三酰甘油的形式储存在脂滴(LDS)中，LDS是内质网衍生的细胞器，由中性脂核组成，周围环绕着装饰着嵌入蛋白质的磷脂单分子层。尽管LD蛋白质组在控制LD功能方面具有明显的重要性，但对其活性和水平的调节机制却知之甚少。翻译后与泛素的结合通过涉及E1、E2和E3酶的酶的级联反应发生，几乎调控着每一个细胞过程。我们的初步发现确定了一条代谢调节的LD泛素化途径，涉及UBXD8介导的p97/VCP的募集和脂解限速酶ATGL催化的LD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作为脂肪酸传感器的假设，该传感器通过靶向脂解所需的酶来稳定LDS以响应代谢信号，从而实现Ub依赖的沉默。具体地说，这项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)，将构建与LD相关的泛素化网络的全面图谱(目标2)，并将识别LD泛素化靶点及其代谢调节 单元格状态(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，以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，我扩大了我的研究培训，包括系统水平的策略、定量蛋白质组学方法和LD功能分析。在该奖项的K99指导阶段，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的公认专家，执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和格莱斯顿心血管疾病研究所的联合环境，这两个环境共同提供了一个充满实验培训、智力增长、合作、网络、职业发展和创新研究机会的环境。我的短期目标是获得一名独立研究员的职位，在获奖的R00期间，我将制定一个强有力的原创研究计划，专注于泛素对LD在健康和疾病中的作用的贡献。我的长期目标是作为一名科学领导者和导师，促进开创性研究，促进对调节脂质代谢的分子机制的基本了解，并最终导致成功开发和实施治疗新陈代谢疾病的有效策略。 公共卫生相关性：与公共健康的相关性：细胞如何储存和动员脂滴中的脂肪酸是一个与肥胖症和糖尿病等代谢性疾病的治疗具有广泛相关性的基本问题。泛素化是一种重要的翻译后修饰，调节细胞中几乎所有已知的过程，但泛素化在脂滴生物学中的作用尚不清楚。该提案中描述的目标将利用最新的技术进步来确定调节脂滴功能的新的泛素化途径的组成部分和下游靶点，并将为开发新的治疗策略奠定基础。

DESCRIPTION (provided by applicant): In order to develop novel therapeutic strategies to combat the increasing epidemic of metabolic diseases (e.g. 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.! PUBLIC HEALTH RELEVANCE: Relevance to public health: How cells store and mobilize fatty acids from lipid droplets is a fundamental question with broad relevance for the treatment of metabolic diseases such as obesity and diabetes. Ubiquitination is an important posttranslational modification that regulates virtually every known process in the cell, but the role of ubiquitination in lipid droplet biology is poorly understood. The aims described in this proposal will utilize recent technological advances to define the components and downstream targets of a new ubiquitination pathway that regulates lipid droplet function, and will lay the foundation for the development of novel therapeutic strategies.!