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

描述（由申请人提供）：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），深入了解脂肪储存和动员的分子机制至关重要。在细胞中，脂肪以三酰甘油的形式储存在脂滴（LDs）中，脂滴是一种内质网衍生的细胞器，由中性脂质核心组成，周围环绕着磷脂单层，磷脂单层由嵌入的蛋白质装饰。尽管LD蛋白质组在控制LD功能中具有明显的重要性，但对调节其活性和水平的机制知之甚少。与泛素的翻译后结合，通过涉及E1，E2和E3酶的酶级联反应发生，几乎调节每一个细胞过程。我们的初步研究结果确定了一个代谢调控的LD泛素化途径，涉及UBXD 8介导的招聘p97/VCP和调节LD营业额催化的ATGL，在脂解的限速酶。这些初步的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检验以下假设，即UBXD 8作为脂肪酸传感器发挥作用，通过靶向脂解所需的酶以实现UB依赖性沉默来稳定LD以响应代谢信号。具体而言，本研究计划将阐明UBXD 8介导的ATGL功能抑制机制（目标1），构建LD相关泛素化网络的综合图谱（目标2），并确定LD泛素化靶点及其通过代谢调控的调节。 单元格的状态（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，我已经扩大了我的研究培训，包括系统级策略，定量蛋白质组学方法和LD功能测定。在这个奖项的指导K99阶段，我将与我的导师罗恩Kopito博士和小罗伯特Farese博士密切合作，在泛素生物学和脂质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计划。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，这两所大学共同提供了一个充满实验培训、智力发展、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个独立研究员的职位，在R 00期间，我将开发一个强大的原创研究计划，重点是泛素对LD在健康和疾病中的作用。我的长期目标是作为一个科学领导者和导师，促进开拓性的研究，促进对调节脂质代谢的分子机制的基本理解，并最终导致成功开发和实施有效的策略来治疗代谢性疾病。

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.!